Antibodies against campylobacter species

ABSTRACT

The instant disclosure provides antibodies and antigen-binding fragments thereof that are specific for Campylobacter and, in certain embodiments, are capable of neutralizing a Campylobacter infection in a subject. In certain embodiments, the antibody or antigen binding fragment comprises an IgA antibody, such as, for example, a secretory IgA antibody. Also provided are pharmaceutical compositions comprising a disclosed antibody or antigen-binding fragment. Methods of using the antibodies, antigen-binding fragments, and compositions to treat or prevent a Campylobacter infection in a subject are also provided. In certain embodiments, recombinant secretory IgA antibodies of the instant disclosure are administered orally to a subject having or at risk of developing a Campylobacter infection.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 470082_406WO_SEQUENCE_LISTING.txt. The text fileis 304 KB, was created on Jul. 16, 2019, and is being submittedelectronically via EFS-Web.

BACKGROUND

Campylobacter is the most common cause of bacterial gastroenteritisworldwide and has recently been added to the World Health Organization(WHO) list of antibiotic resistant bacteria that pose a potential globalthreat to human health (see, e.g., “WHO publishes list of bacteria forwhich new antibiotics are urgently needed”, World Health Organizationnews release, Feb. 27 2017;who.int/en/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed).Campylobacter species (C. jejuni and C. coli) are a significant cause oftraveler's diarrhea in developed countries and a major cause oflife-threatening acute watery diarrhea in children under the age of 2 indeveloping countries. Currently, there are no vaccines approved toprevent Campylobacteriosis. Rehydration is the main form of therapy, andalthough antibiotics have been shown to be beneficial in severeinfections, they are often not recommended to avoid the rapiddevelopment of resistance.

Accordingly, new therapies for preventing or treating Campylobacterinfections are needed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cross-reactivity with murine microbiota and breadth ofCampylobacter (“Campy”)-reactive rSIgA of the present disclosure.

FIG. 2 shows the design of a bacterial motility assay examining theability of Campy-reactive rSIgA of the present disclosure to limitbacteria motility.

FIG. 3 shows results from the experiment shown in FIG. 2. Culture plateswere as follows: (left to right)=mock infected; no mAb; ctrl rSIgA;Campy-reactive rSIgA.

FIG. 4A shows FACS analysis of human IgA-coated bacteria in the stoolsof C57BL/6 mice that received a prophylactic dose of Campy-reactiverSIgA 1 h prior to infection with 10⁹ CFU of Campylobacter jejuni strain81-176, followed by a second dose of rSIgA at 6 h p.i. Stool sampleswere taken at 4 h, 6 h, and 9 h p.i., as indicated.

FIG. 4B shows anti-HuIgA-coated bacteria in the stools of uninfected (L)versus infected (R) mice.

FIGS. 5A and 5B show Campylobacter shedding in treated and untreatedanimals at (A) 24 hours and (B) 72 hours post-infection.

FIG. 6 shows ELISA quantification of Lipocalin-2 at 24, 48 and 72 hourspost-infection in animal stools.

FIGS. 7A-7E show in vitro characterization of exemplary antibodies CAA1and CCG4 of the present disclosure (expressed as rSIgA) binding to FliD.(A) Binding of CAA1, CCG4 and HGN194 rSIgA to coated recombinant FliD asmeasured by ELISA. Serial dilutions of the three mAbs were incubated for1 h at RT with FliD pre-coated 96 well ELISA plates. Detection wasperformed using a biotinylated anti-human SC antibody followed byincubation with Streptavidin-AP. (B) Cross-competition studies performedby bio-layer interferometry (BLI). FliD antigen was immobilized on APSsensors and then incubated with CAA1 prior to association with CGG4,CAA1 or PBS with 1% BSA. (C) Western blot analysis of CAA1, CCG4 andHGN194 rSIgA binding to FliD antigen (70 KDa) under reducing anddenaturing conditions. (D) Representative histograms of the in vitrobinding of the indicated mAbs against pure culture of C. jejuni and C.coli. One representative experiment out of three is shown. (E) Bindingof CAA1, CCG4 and HGN194 rSIgA2 to C. jejuni as observed in confocalmicroscopy. Bacteria were stained using Syto BC, whereas the mAbs weredetected using anti-human IgA AF647 conjugated.

FIGS. 8A-8D show that C57BL/6 just-weaned mice are highly sensitive toC. jejuni infection. (A-C) C57BL/6 mice at 12, 21 and 56 days of agewere orally infected with 10⁸ CFU of C. jejuni. (A) Bacteria loads(CFU), and (B) Lipocalin 2 (LCN2) in the stools of infected animals weredetermined at 6 days post-infection. (C) Representative H&E sections ofthe caecum from infected mice and statistical analysis ofhistopathological scores at 6 days post-infection. White arrows:submucosal inflammation; white asterisk: crypt hyperplasia withdecreased number of goblet cells; black asterisk: epithelialdesquamation; black arrows: mucosal inflammation. Scale bar: 200 (D)Quantification of fecal IgA concentrations in C57BL/6 mice at 12, 21 and56 days of age. Dots represent individual mice and results are shown as±SEM. Mann-Whitney test (A-D) was used. *p<0.05, **p<0.01. Onerepresentative experiment out of two is shown.

FIGS. 9A-9D show prophylactic activity of orally administered CAA1 andCCG4 rSIgA2 against C. jejuni infection in just-weaned mice. (A-D) Twohours prior to infection with 10⁸ cfu of C. jejuni, 21-day-old C57BL/6mice were orally administered by gavage with 200 μg of the indicatedmAbs in PBS. (A) Fecal bacterial loads (CFU) at 24 h, 48 h and 72 h postC. jejuni infection were determined. (B) Lipocalin-2 (LCN) levels in thestools, (C) statistical analysis of polymorphonucleated (PMN) cellinfiltrates gated as Gr1⁺CD11b⁺, and (D) histopathological score in thecaecum were determined at 72 h post infection in the different treatmentconditions. Dots represent individual mice and results are shown as±SEM. Mann-Whitney test (A-D) was used. *p<0.05, **p<0.01, ***p<0.001.One representative experiment out of at least two is shown.

FIGS. 10A-10D show that CAA1 SIgA1 and SIgA2 have similar prophylacticactivity against C. jejuni infection. (A-D) Two hours prior to infectionwith 10⁸ CFU of C. jejuni, 21-day-old C57BL/6 mice were orallyadministered via gavage with 200 of CAA1 as rSIgA1 or rSIgA2. (A)Quantification of the bacterial load (CFU) in the stools of the animalsat 24 h, 48 h and 72 h post-infection. (B) Representative dot plot andrelative quantification of polymorphonucleated cells infiltrated in thecaecum gated as Gr1⁺CD11b⁺. (C) Quantification of Lipocalin-2 (LCN) inthe stools, and (D) statistical analysis of histopathological score inthe caecum at 72 h post infection in the different treatment conditions.Dots represent individual mice and results are shown as ±SEM.Mann-Whitney test (A-D) was used. *p<0.05, **p<0.01. One representativeexperiment out of at least two is shown.

FIGS. 11A-11D show that conversion to IgG reduces oral CAA1 prophylacticactivity against C. jejuni infection. (A-D) 2 h prior to infection with10⁸ CFU of C. jejuni, 21-day-old C57BL/6 mice were orally administered,via gavage, 200 μg of CAA1 as rSIgA2 or rIgG1. (A) Quantification of thebacterial load (CFU) in the stools of the animals at 24 h, 48 h and 72 hpost-infection. (B) Representative dot plot and relative quantificationof polymorphonucleated cells infiltrated in the caecum gated as Gr1⁺CD1lb⁺. (C) Quantification of Lipocalin-2 (LCN) in the stools, and (D)statistical analysis of histopathological score in the caecum at 72 hpost infection in the different treatment conditions. Dots representindividual mice and results are shown as ±SEM. Mann-Whitney test (A-D)was used. *p<0.05, **p<0.01. One representative experiment out of atleast two is shown.

FIG. 12 shows analysis of FliD amino acid sequence conservation. FliDamino acid sequences from C. jejuni and C. coli isolates were retrievedfrom GenBank and analyzed using CLC Main Workbench software (Qiagen).The height of each letter in the sequence logo represents the level ofconservation of that amino acid at the specific site. The consensussequence for FliD is shown at top.

FIG. 13 shows frequency of FliD-reactive IgA⁺ and IgG⁺ memory B cellsfrom different tonsillar samples. Analysis of reactivity against FliDantigen of the IgA⁺ (upper panel) and IgG⁺ (lower panel) memory B cellrepertoire for different tonsillar samples is shown.

FIGS. 14A-14C show cross-reactivity of FliD-reactive mAbs with themurine microbiota and persistence in caecum of C57BL/6 just weaned mice.(A) Representative histograms of the specific binding of the indicatedmAbs against fecal microbiota of mice mock infected or infected with C.jejuni or C. coli. One representative experiment out of three is shown.(B-C) Pharmacokinetics evaluation by ELISA of HGN194 (B) rSIgA and (C)rIgG antibody at the indicated time points in the different mouseintestinal sub-compartments. One representative experiment out of at twois shown.

FIGS. 15A-15D provide further data showing prophylactic activity of CAA1rSIgA at different C. jejuni infection doses. (A-B) Quantification ofthe fecal bacterial load (CFU) in 21-day-old C57BL/6 mice administeredvia gavage with 200 μg of CAA1 rSIgA2 as measured at 24, 72 and 120 hpost-infection with 10⁷ or 10⁹ CFU of C. jejuni. (C-D) Quantification offecal lipocalin-2 (LCN) in 21-day-old C57BL/6 mice administered viagavage with 200 μg of CAA1 rSIgA2 as measured at 120 h post-infectionwith 10⁷ or 10⁹ CFU of C. jejuni. Dots represent individual mice andresults are shown as ±SEM. Mann-Whitney test (A-D) was used. *p<0.05,**p<0.01. One representative experiment out of at least two is shown.

FIGS. 16A-16D show that IgA isotype switch affects Igase sensitivity,but not FliD affinity or specificity. (A) Denaturing non-reducing gel ofCAA1 rSIgA1 and rSIgA2 incubated over-night (18 hours) with PBS or withrIgA protease (IgAse Pro-Pro-Y-Pro) from Neisseria gonorrhoeae. (1:SIgA1+PBS; 2: SIgA2+PBS; 3: SIgA1+Igase; 4: SIgA2+Igase). (B) Binding ofrSlgA1 and rSIgA2 CAA1 to FliD, as measured by ELISA. Serial dilutionsof the mAbs were incubated for 1 h at RT with FliD pre-coated 96 wellELISA plates. Detection was performed using a biotinylated anti-human SCantibody followed by incubation with Streptavidin-AP. (C) Representativehistograms of the in vitro specific binding of the indicated mAbsagainst pure culture of C. jejuni and C. coli. One representativeexperiment out of three is shown. (D) Representative histograms of CAA1rSlgA1 and rSIgA2 binding to the fecal microbiota of not infectedC57BL/6 just-weaned mice.

FIGS. 17A-17D show that conversion to IgG format reduces oral CCG4prophylactic activity against C. jejuni infection. (A-D) 2 h prior toinfection with 10⁸ CFU of C. jejuni, 21-day-old C57BL/6 mice were orallyadministered via gavage with 200 μg of CCg4 as rSIgA2 or rIgG1. (A)Quantification of the bacterial load (CFU) in the stools of the animalsat 24 and 72 h post-infection. (B) Representative dot plot and relativequantification of polymorphonucleated cells infiltrated in the caecum at72 h post-infection. (C) Quantification of Lipocalin-2 (LCN) in thestools, and (D) statistical analysis of histopathological score in thecaecum at 72 h post infection in the different treatment conditions.Dots represent individual mice and results are shown as ±SEM.Mann-Whitney test (A-D) was used. *p<0.05, **p<0.01. One representativeexperiment out of two is shown.

DETAILED DESCRIPTION

Provided herein are antibodies and antigen-binding fragments specificfor Campylobacter, compositions comprising the same, and methods ofusing the antibodies and compositions to treat (e.g., reduce, delay,eliminate, or prevent) a Campylobacter infection in a subject. In someembodiments, an antibody of the present disclosure comprises an IgAmolecule, such as a dimeric IgA molecule. In certain embodiments, an IgAantibody of the present disclosure is provided in a secretory form(SIgA), as described herein. Administration of antibodies andantigen-binding fragments of the present disclosure, e.g., via oraldelivery of a presently disclosed SIgA, can treat infection byCampylobacter, such as Campylobacter species associated with severeneonatal gastroenteritis.

By way of background, Campylobacter is an established cause of diarrheaworldwide and has recently been added to the WHO list of bacteria whoseantibiotic resistance might pose a global threat to human health (WorldHealth Organization (WHO), 2017). Campylobacter's epidemiology differsbetween high-income countries, where the encounter with the bacteria issporadic, and low- and middle-income countries, in which the infectionis almost universal in early childhood, and is a major cause oflife-threatening acute watery diarrhea in infants (Riddle and Guerry,Vaccine, 34:2903-2906 (2016)).

Considered as a leading zoonosis, Campylobacter infection is mainlyassociated with the consumption of contaminated undercooked animal meat(poultry being the primary bacteria reservoir), water or unpasteurizedmilk (Kaakoush et al., Clin. Microbiol. Rev., 28:687-720 (2015)).Campylobacter jejuni and C. coli are major causes of Campylobacterenteritis in humans (Man, Nat. Rev. Gastroenterol. Hepatol., 8:669-685(2011)).

Campylobacteriosis typically results in an acute, gastrointestinalillness characterized by watery or bloody diarrhea, fever, weight loss,and cramps that last on average 6 days Kaakoush et al., Clin. Microbiol.Rev., 28:687-720 (2015); World Health Organization (WHO) (2013)). Severedehydration associated with Campylobacter enteritis represents asignificant cause of death among newborns and children, particularly indeveloping countries (Platts-Mills et al., Lancet Glob. Health.,3:e564-75 (2015)). Furthermore, C. jejuni infection has beenconsistently linked with the onset of autoimmune conditions such asGuillain-Barré Syndrome (GB S) (Islam et al., PLoS One, 7: e43976(2012); Yuki et al., Proc. Natl. Acad. Sci. U.S.A., 101:11404-11409(2004)) and Inflammatory Bowel Disease (IBD) (Gradel et al.,Gastroenterology, 137:495-501 (2009)).

Flagellum-mediated motility is thought to be important forCampylobacter's virulence and pathogenicity, as shown in bothexperimental animal models and in human healthy volunteer studies (Blacket al., J. Infect. Dis., 157:472-479 (1988); Morooka et al., J. Gen.Microbiol., 131:1973-1980 (1985)). But, flagellin (FlaA), the majorconstituent of the flagellum, does not present a high level ofconservation even within the same C. jejuni species, and its heavyglycosylation pattern varies greatly depending on the strain and growthphase (Parkhill et al., Nature, 403:665-668 (2000); Thibault et al., J.Biol. Chem., 276:34862-34870 (2001)). A recombinant non-glycosylatedform of C. jejuni flagellin was shown to be poorly immunogenic inclinical trials (Riddle and Guerry, Vaccine, 34:2903-2906 (2016)),making FlaA a challenging target for therapy. Moreover, the possibilityto use C. jejuni in a vaccine has been limited by the risk of GBSdevelopment associated with ganglioside mimicry of bacteriallipo-oligosaccharide (LOS) (Riddle and Guerry, Vaccine, 34:2903-2906(2016)).

Due to these shortcomings, there are currently no vaccines approved by aglobal regulatory authority to prevent Campylobacter infection.Rehydration is the main form of therapy, and while antibiotics have beenshown to be beneficial in severe infections, they are often notrecommended due to the rapid development of antibiotic resistance. Evenin the case of recovery from the infection, the continuous exposure ofinfants in low-income countries to intestinal pathogens, includingCampylobacter, has been linked to environmental enteropathy(EE)/environmental enteric dysfunction (EED), a subclinical chronicinflammation of the small intestine associated with malabsorption ofnutrients, growth faltering, impaired cognitive development, changes inmicrobiota, and reduced responsiveness to oral vaccination (Watanabe andPetri, EBioMedicine, 10:25-32 (2016)).

The present disclosure provides antibodies and antigen-binding fragmentsthat bind to the Campylobacter flagellar-capping protein FliD.Antibodies according to the present disclosure advantageously limitmotility of Campylobacter and, in an animal model of Campylobacterinfection described in this disclosure, are capable of boostingCampylobacter clearance infection, significantly reducing the levels ofinflammation markers associated with epithelial damage andpolymorphonuclear (PMN) cells infiltration.

Also provided herein are compositions that comprise a CampylobacterFliD-specific antibody or antigen-binding fragment of the presentdisclosure, polynucleotides that encode the antibody or antigen-bindingfragment, vectors that contain the polynucleotide, and host cells thatexpress the antibody or antigen-binding fragment, and/or comprise orcontain a polynucleotide or vector of the present disclosure. Methodsand uses are also provided for treating a Campylobacter infection and/orfor reducing an associated symptom.

Also provided are non-human animal models for studying Campylobacterinfection.

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, the term “about” means±20% of theindicated range, value, or structure, unless otherwise indicated. Itshould be understood that the terms “a” and “an” as used herein refer to“one or more” of the enumerated components. The use of the alternative(e.g., “or”) should be understood to mean either one, both, or anycombination thereof of the alternatives. As used herein, the terms“include,” “have,” and “comprise” are used synonymously, which terms andvariants thereof are intended to be construed as non-limiting.

“Optional” or “optionally” means that the subsequently describedelement, component, event, or circumstance may or may not occur, andthat the description includes instances in which the element, component,event, or circumstance occurs and instances in which they do not.

In addition, it should be understood that the individual constructs, orgroups of constructs, derived from the various combinations of thestructures and subunits described herein, are disclosed by the presentapplication to the same extent as if each construct or group ofconstructs was set forth individually. Thus, selection of particularstructures or particular subunits is within the scope of the presentdisclosure.

The term “consisting essentially of” is not equivalent to “comprising”and refers to the specified materials or steps of a claim, or to thosethat do not materially affect the basic characteristics of a claimedsubject matter. For example, a protein domain, region, or module (e.g.,a binding domain, hinge region, or linker) or a protein (which may haveone or more domains, regions, or modules) “consists essentially of” aparticular amino acid sequence when the amino acid sequence of a domain,region, module, or protein includes extensions, deletions, mutations, ora combination thereof (e.g., amino acids at the amino- orcarboxy-terminus or between domains) that, in combination, contribute toat most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) ofthe length of a domain, region, module, or protein and do notsubstantially affect (i.e., do not reduce the activity by more than 50%,such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) theactivity of the domain(s), region(s), module(s), or protein (e.g., thetarget binding affinity of a binding protein).

As used herein, “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α-carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refer tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

As used herein, “mutation” refers to a change in the sequence of anucleic acid molecule or polypeptide molecule as compared to a referenceor wild-type nucleic acid molecule or polypeptide molecule,respectively. A mutation can result in several different types of changein sequence, including substitution, insertion or deletion ofnucleotide(s) or amino acid(s).

A “conservative substitution” refers to amino acid substitutions that donot significantly affect or alter binding characteristics of aparticular protein. Generally, conservative substitutions are ones inwhich a substituted amino acid residue is replaced with an amino acidresidue having a similar side chain. Conservative substitutions includea substitution found in one of the following groups: Group 1: Alanine(Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T);Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3:Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg orR), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile orI), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); andGroup 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trpor W). Additionally or alternatively, amino acids can be grouped intoconservative substitution groups by similar function, chemicalstructure, or composition (e.g., acidic, basic, aliphatic, aromatic, orsulfur-containing). For example, an aliphatic grouping may include, forpurposes of substitution, Gly, Ala, Val, Leu, and Ile. Otherconservative substitutions groups include: sulfur-containing: Met andCysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar,negatively charged residues and their amides: Asp, Asn, Glu, and Gln;polar, positively charged residues: His, Arg, and Lys; large aliphatic,nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromaticresidues: Phe, Tyr, and Trp. Additional information can be found inCreighton (1984) Proteins, W.H. Freeman and Company.

As used herein, “protein” or “polypeptide” refers to a polymer of aminoacid residues. Proteins apply to naturally occurring amino acidpolymers, as well as to amino acid polymers in which one or more aminoacid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid and non-naturally occurring amino acidpolymers. Variants of proteins, peptides, and polypeptides of thisdisclosure are also contemplated. In certain embodiments, variantproteins, peptides, and polypeptides comprise or consist of an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acidsequence of a defined or reference amino acid sequence as describedherein.

“Nucleic acid molecule” or “polynucleotide” or “polynucleic acid” refersto a polymeric compound including covalently linked nucleotides, whichcan be made up of natural subunits (e.g., purine or pyrimidine bases) ornon-natural subunits (e.g., morpholine ring). Purine bases includeadenine, guanine, hypoxanthine, and xanthine, and pyrimidine basesinclude uracil, thymine, and cytosine. Nucleic acid molecules includepolyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viralgenomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA),which includes cDNA, genomic DNA, and synthetic DNA, either of which maybe single or double stranded. If single-stranded, the nucleic acidmolecule may be the coding strand or non-coding (anti-sense) strand. Anucleic acid molecule encoding an amino acid sequence includes allnucleotide sequences that encode the same amino acid sequence. Someversions of the nucleotide sequences may also include intron(s) to theextent that the intron(s) would be removed through co- orpost-transcriptional mechanisms. In other words, different nucleotidesequences may encode the same amino acid sequence as the result of theredundancy or degeneracy of the genetic code, or by splicing.

Variants of nucleic acid molecules of this disclosure are alsocontemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%,85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identicala nucleic acid molecule of a defined or reference polynucleotide asdescribed herein, or that hybridize to a polynucleotide under stringenthybridization conditions of 0.015M sodium chloride, 0.0015M sodiumcitrate at about 65-68° C. or 0.015M sodium chloride, 0.0015M sodiumcitrate, and 50% formamide at about 42° C. Nucleic acid moleculevariants retain the capacity to encode a binding domain thereof having afunctionality described herein, such as binding a target molecule.

“Percent sequence identity” refers to a relationship between two or moresequences, as determined by comparing the sequences. Preferred methodsto determine sequence identity are designed to give the best matchbetween the sequences being compared. For example, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment). Further, non-homologous sequences may bedisregarded for comparison purposes. The percent sequence identityreferenced herein is calculated over the length of the referencesequence, unless indicated otherwise. Methods to determine sequenceidentity and similarity can be found in publicly available computerprograms. Sequence alignments and percent identity calculations may beperformed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, orBLASTX). The mathematical algorithm used in the BLAST programs can befound in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Withinthe context of this disclosure, it will be understood that wheresequence analysis software is used for analysis, the results of theanalysis are based on the “default values” of the program referenced.“Default values” mean any set of values or parameters which originallyload with the software when first initialized.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring nucleic acid orpolypeptide present in a living animal is not isolated, but the samenucleic acid or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Such nucleicacid could be part of a vector and/or such nucleic acid or polypeptidecould be part of a composition (e.g., a cell lysate), and still beisolated in that such vector or composition is not part of the naturalenvironment for the nucleic acid or polypeptide.

The term “gene” means the segment of DNA or RNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (e.g., 5′ untranslated region (UTR) and 3′ UTR) as well asintervening sequences (introns) between individual coding segments(exons).

A “functional variant” refers to a polypeptide or polynucleotide that isstructurally similar or substantially structurally similar to a parentor reference compound of this disclosure, but differs slightly incomposition (e.g., one base, atom or functional group is different,added, or removed), such that the polypeptide or encoded polypeptide iscapable of performing at least one function of the parent polypeptidewith at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activityof the parent polypeptide. In other words, a functional variant of apolypeptide or encoded polypeptide of this disclosure has “similarbinding,” “similar affinity” or “similar activity” when the functionalvariant displays no more than a 50% reduction in performance in aselected assay as compared to the parent or reference polypeptide, suchas an assay for measuring binding affinity (e.g., Biacore® or tetramerstaining measuring an association (Ka) or a dissociation (KD) constant).

As used herein, a “functional portion” or “functional fragment” refersto a polypeptide or polynucleotide that comprises only a domain, portionor fragment of a parent or reference compound, and the polypeptide orencoded polypeptide retains at least 50% activity associated with thedomain, portion or fragment of the parent or reference compound,preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, orprovides a biological benefit (e.g., effector function). A “functionalportion” or “functional fragment” of a polypeptide or encodedpolypeptide of this disclosure has “similar binding” or “similaractivity” when the functional portion or fragment displays no more thana 50% reduction in performance in a selected assay as compared to theparent or reference polypeptide (preferably no more than 20% or 10%, orno more than a log difference as compared to the parent or referencewith regard to affinity).

As used herein, the term “engineered,” “recombinant,” or “non-natural”refers to an organism, microorganism, cell, nucleic acid molecule, orvector that includes at least one genetic alteration or has beenmodified by introduction of an exogenous or heterologous nucleic acidmolecule, wherein such alterations or modifications are introduced bygenetic engineering (i.e., human intervention). Genetic alterationsinclude, for example, modifications introducing expressible nucleic acidmolecules encoding functional RNA, proteins, fusion proteins or enzymes,or other nucleic acid molecule additions, deletions, substitutions, orother functional disruption of a cell's genetic material. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a polynucleotide, gene, oroperon.

As used herein, “heterologous” or “non-endogenous” or “exogenous” refersto any gene, protein, compound, nucleic acid molecule, or activity thatis not native to a host cell or a subject, or any gene, protein,compound, nucleic acid molecule, or activity native to a host cell or asubject that has been altered. Heterologous, non-endogenous, orexogenous includes genes, proteins, compounds, or nucleic acid moleculesthat have been mutated or otherwise altered such that the structure,activity, or both is different as between the native and altered genes,proteins, compounds, or nucleic acid molecules. In certain embodiments,heterologous, non-endogenous, or exogenous genes, proteins, or nucleicacid molecules (e.g., receptors, ligands, etc.) may not be endogenous toa host cell or a subject, but instead nucleic acids encoding such genes,proteins, or nucleic acid molecules may have been added to a host cellby conjugation, transformation, transfection, electroporation, or thelike, wherein the added nucleic acid molecule may integrate into a hostcell genome or can exist as extra-chromosomal genetic material (e.g., asa plasmid or other self-replicating vector). The term “homologous” or“homolog” refers to a gene, protein, compound, nucleic acid molecule, oractivity found in or derived from a host cell, species, or strain. Forexample, a heterologous or exogenous polynucleotide or gene encoding apolypeptide may be homologous to a native polynucleotide or gene andencode a homologous polypeptide or activity, but the polynucleotide orpolypeptide may have an altered structure, sequence, expression level,or any combination thereof. A non-endogenous polynucleotide or gene, aswell as the encoded polypeptide or activity, may be from the samespecies, a different species, or a combination thereof.

In certain embodiments, a nucleic acid molecule or portion thereofnative to a host cell will be considered heterologous to the host cellif it has been altered or mutated, or a nucleic acid molecule native toa host cell may be considered heterologous if it has been altered with aheterologous expression control sequence or has been altered with anendogenous expression control sequence not normally associated with thenucleic acid molecule native to a host cell. In addition, the term“heterologous” can refer to a biological activity that is different,altered, or not endogenous to a host cell. As described herein, morethan one heterologous nucleic acid molecule can be introduced into ahost cell as separate nucleic acid molecules, as a plurality ofindividually controlled genes, as a polycistronic nucleic acid molecule,as a single nucleic acid molecule encoding a fusion protein, or anycombination thereof. When

As used herein, the term “endogenous” or “native” refers to apolynucleotide, gene, protein, compound, molecule, or activity that isnormally present in a host cell or a subject.

The term “expression”, as used herein, refers to the process by which apolypeptide is produced based on the encoding sequence of a nucleic acidmolecule, such as a gene. The process may include transcription,post-transcriptional control, post-transcriptional modification,translation, post-translational control, post-translationalmodification, or any combination thereof. An expressed nucleic acidmolecule is typically operably linked to an expression control sequence(e.g., a promoter).

The term “operably linked” refers to the association of two or morenucleic acid molecules on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., the coding sequence isunder the transcriptional control of the promoter). “Unlinked” meansthat the associated genetic elements are not closely associated with oneanother and the function of one does not affect the other.

As described herein, more than one heterologous nucleic acid moleculecan be introduced into a host cell as separate nucleic acid molecules,as a plurality of individually controlled genes, as a polycistronicnucleic acid molecule, as a single nucleic acid molecule encoding afusion protein, or any combination thereof. When two or moreheterologous nucleic acid molecules are introduced into a host cell, itis understood that the two or more heterologous nucleic acid moleculescan be introduced as a single nucleic acid molecule (e.g., on a singlevector), on separate vectors, integrated into the host chromosome at asingle site or multiple sites, or any combination thereof. The number ofreferenced heterologous nucleic acid molecules or protein activitiesrefers to the number of encoding nucleic acid molecules or the number ofprotein activities, not the number of separate nucleic acid moleculesintroduced into a host cell.

The term “construct” refers to any polynucleotide that contains arecombinant nucleic acid molecule (or, when the context clearlyindicates, a fusion protein of the present disclosure). A(polynucleotide) construct may be present in a vector (e.g., a bacterialvector, a viral vector) or may be integrated into a genome. A “vector”is a nucleic acid molecule that is capable of transporting anothernucleic acid molecule. Vectors may be, for example, plasmids, cosmids,viruses, a RNA vector or a linear or circular DNA or RNA molecule thatmay include chromosomal, non-chromosomal, semi-synthetic or syntheticnucleic acid molecules. Vectors of the present disclosure also includetransposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al.,Mol. Ther. 8:108, 2003: Matés et al., Nat. Genet. 41:753, 2009).Exemplary vectors are those capable of autonomous replication (episomalvector), capable of delivering a polynucleotide to a cell genome (e.g.,viral vector), or capable of expressing nucleic acid molecules to whichthey are linked (expression vectors).

As used herein, “expression vector” or “vector” refers to a DNAconstruct containing a nucleic acid molecule that is operably linked toa suitable control sequence capable of effecting the expression of thenucleic acid molecule in a suitable host. Such control sequences includea promoter to effect transcription, an optional operator sequence tocontrol such transcription, a sequence encoding suitable mRNA ribosomebinding sites, and sequences which control termination of transcriptionand translation. The vector may be a plasmid, a phage particle, a virus,or simply a potential genomic insert. Once transformed into a suitablehost, the vector may replicate and function independently of the hostgenome, or may, in some instances, integrate into the genome itself ordeliver the polynucleotide contained in the vector into the genomewithout the vector sequence. In the present specification, “plasmid,”“expression plasmid,” “virus,” and “vector” are often usedinterchangeably.

The term “introduced” in the context of inserting a nucleic acidmolecule into a cell, means “transfection”, “transformation,” or“transduction” and includes reference to the incorporation of a nucleicacid molecule into a eukaryotic or prokaryotic cell wherein the nucleicacid molecule may be incorporated into the genome of a cell (e.g.,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

In certain embodiments, polynucleotides of the present disclosure may beoperatively linked to certain elements of a vector. For example,polynucleotide sequences that are needed to effect the expression andprocessing of coding sequences to which they are ligated may beoperatively linked. Expression control sequences may include appropriatetranscription initiation, termination, promoter, and enhancer sequences;efficient RNA processing signals such as splicing and polyadenylationsignals; sequences that stabilize cytoplasmic mRNA; sequences thatenhance translation efficiency (i.e., Kozak consensus sequences);sequences that enhance protein stability; and possibly sequences thatenhance protein secretion. Expression control sequences may beoperatively linked if they are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

In certain embodiments, the vector comprises a plasmid vector or a viralvector (e.g., a lentiviral vector or a γ-retroviral vector). Viralvectors include retrovirus, adenovirus, parvovirus (e.g.,adeno-associated viruses), coronavirus, negative strand RNA viruses suchas ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabiesand vesicular stomatitis virus), paramyxovirus (e.g., measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox, andcanarypox). Other viruses include, for example, Norwalk virus,togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, andhepatitis virus. Examples of retroviruses include avianleukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses,HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: Theviruses and their replication, In Fundamental Virology, Third Edition,B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia,1996).

“Retroviruses” are viruses having an RNA genome, which isreverse-transcribed into DNA using a reverse transcriptase enzyme, thereverse-transcribed DNA is then incorporated into the host cell genome.“Gammaretrovirus” refers to a genus of the retroviridae family. Examplesof gammaretroviruses include mouse stem cell virus, murine leukemiavirus, feline leukemia virus, feline sarcoma virus, and avianreticuloendotheliosis viruses.

“Lentiviral vectors” include HIV-based lentiviral vectors for genedelivery, which can be integrative or non-integrative, have relativelylarge packaging capacity, and can transduce a range of different celltypes. Lentiviral vectors are usually generated following transienttransfection of three (packaging, envelope, and transfer) or moreplasmids into producer cells. Like HIV, lentiviral vectors enter thetarget cell through the interaction of viral surface glycoproteins withreceptors on the cell surface. On entry, the viral RNA undergoes reversetranscription, which is mediated by the viral reverse transcriptasecomplex. The product of reverse transcription is a double-strandedlinear viral DNA, which is the substrate for viral integration into theDNA of infected cells.

In certain embodiments, the viral vector can be a gammaretrovirus, e.g.,Moloney murine leukemia virus (MLV)-derived vectors. In otherembodiments, the viral vector can be a more complex retrovirus-derivedvector, e.g., a lentivirus-derived vector. HIV-1-derived vectors belongto this category. Other examples include lentivirus vectors derived fromHIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus(ovine lentivirus). Methods of using retroviral and lentiviral viralvectors and packaging cells for transducing mammalian host cells withviral particles containing transgenes are known in the art and have beenprevious described, for example, in: U.S. Pat. No. 8,119,772; Walchli etal., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005;Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther.18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009.Retroviral and lentiviral vector constructs and expression systems arealso commercially available. Other viral vectors also can be used forpolynucleotide delivery including DNA viral vectors, including, forexample adenovirus-based vectors and adeno-associated virus (AAV)-basedvectors; vectors derived from herpes simplex viruses (HSVs), includingamplicon vectors, replication-defective HSV and attenuated HSV (Kriskyet al., Gene Ther. 5:1517, 1998).

Other vectors that can be used with the compositions and methods of thisdisclosure include those derived from baculoviruses and α-viruses.(Jolly, D J. 1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed.The Development of Human Gene Therapy. New York: Cold Spring HarborLab), or plasmid vectors (such as sleeping beauty or other transposonvectors).

When a viral vector genome comprises a plurality of polynucleotides tobe expressed in a host cell as separate transcripts, the viral vectormay also comprise additional sequences between the two (or more)transcripts allowing for bicistronic or multicistronic expression.Examples of such sequences used in viral vectors include internalribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, orany combination thereof.

As used herein, the term “host” refers to a cell or microorganismtargeted for genetic modification with a heterologous nucleic acidmolecule to produce a polypeptide of interest (e.g., an antibody of thepresent disclosure).

A host cell may include any individual cell or cell culture which mayreceive a vector or the incorporation of nucleic acids or expressproteins. The term also encompasses progeny of the host cell, whethergenetically or phenotypically the same or different. Suitable host cellsmay depend on the vector and may include mammalian cells, animal cells,human cells, simian cells, insect cells, yeast cells, and bacterialcells. These cells may be induced to incorporate the vector or othermaterial by use of a viral vector, transformation via calcium phosphateprecipitation, DEAE-dextran, electroporation, microinjection, or othermethods. See, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).

As used herein, “flagellar-capping protein”, also referred to as “FliD”,and “hook-associated protein 2 (HAP2)”, is an approximately 70 kDaprotein with high sequence conservation across the C. jejuni and C. colispecies (Chintoan-Uta et al., Vaccine, 34:1739-1743 (2016)) (e.g., FIG.12; SEQ ID NOs:43-95). Without wishing to be bound by theory, it isbelieved that Campylobacter FliD oligomers form a cap protein complexlocated at the tip of the flagellum, which controls the distal growth ofthe filament by regulating the assembly of the flagellin molecules. Dueto its functional role in filament elongation, FliD-deficient mutantsexhibit defects in bacterial motility (Song et al., J. Mol. Biol.,429:847-857 (2017)). FliD has been proposed to be involved in celladherence (Freitag et al., Cell. Microbiol., (2017)) and immunogenicityin chickens during natural infection.

“Antigen” or “Ag”, as used herein, refers to an immunogenic moleculethat provokes an immune response. This immune response may involveantibody production, activation of specific immunologically-competentcells, activation of complement, antibody dependent cytotoxicicity, orany combination thereof. An antigen (immunogenic molecule) may be, forexample, a peptide, glycopeptide, polypeptide, glycopolypeptide,polynucleotide, polysaccharide, lipid, or the like. It is readilyapparent that an antigen can be synthesized, produced recombinantly, orderived from a biological sample. Exemplary biological samples that cancontain one or more antigens include tissue samples, stool samples,cells, biological fluids, or combinations thereof. Antigens can beproduced by cells that have been modified or genetically engineered toexpress an antigen. Antigens can also be present in a Campylobacter;e.g., a FliD protein or portion thereof.

The term “epitope” or “antigenic epitope” includes any molecule,structure, amino acid sequence, or protein determinant that isrecognized and specifically bound by a cognate binding molecule, such asan immunoglobulin, or other binding molecule, domain, or protein.Epitopic determinants generally contain chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three dimensional structural characteristics, as wellas specific charge characteristics. Where an antigen is or comprises apeptide or protein, the epitope can be comprised of consecutive aminoacids (e.g., a linear epitope), or can be comprised of amino acids fromdifferent parts or regions of the protein that are brought intoproximity by protein folding (e.g., a discontinuous or conformationalepitope), or non-contiguous amino acids that are in close proximityirrespective of protein folding.

Antibodies, Antigen-Binding Fragments, and Compositions

In one aspect, the present disclosure provides an isolated antibody, oran antigen-binding fragment thereof, that is specific for aCampylobacter flagellum capping protein (FliD) epitope. In certainembodiments, the epitope is a conformational epitope. In otherembodiments, the epitope is a linear epitope.

An antibody or antigen-binding fragment of the present disclosure is“specific for” a FliD epitope or antigen, meaning that it associateswith or unites with the epitope or antigen comprising the epitope, whilenot significantly associating or uniting with any other molecules orcomponents in a sample. In certain embodiments, an antibody orantigen-binding fragment of the present disclosure associates with orunites (e.g., binds) to FliD, while not significantly associating withother molecules or components (e.g., other antigens or potentialantigens, including other Campylobacter proteins) present in a sample.In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure that is specific for FliD is capable of binding tothe FliD epitope with an EC50 of less than about 0.1 μg/mL, or less thanabout 0.05 μg/mL, or less than about 0.03 μg/mL, as measured by ELISA.In certain embodiments, the antibody or antigen-binding fragment iscapable of binding to the FliD epitope with an EC50 of about 0.03 μg/mL,or about 0.025 μg/mL, or about 0.020 μg/mL.

In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure is capable of binding to the FliD epitope with anEC50 of less than about 0.1 μg/mL (i.e., less than about 0.1, 0.09,0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.025, or 0.02 μg/mL, or less), asmeasured by ELISA (e.g., with a readout of OD 450 nm). In certainembodiments, an antibody or antigen-binding fragment of the presentdisclosure is capable of binding to the FliD epitope with an EC50 ofless than about 0.05 μg/mL, or less than about 0.03 μg/mL, as measuredby ELISA (e.g., with a readout of OD 450 nm).

An exemplary assay for measuring EC50 of an antibody or antigen-bindingfragment for FliD includes incubating the antibody or antigen-bindingfragment for about 1 h at RT with FliD-pre-coated 96-well ELISA plates,and then performing detection using a biotinylated anti-Ig SC antibodyfollowed by incubation with Streptavidin-AP.

As used herein, “specifically binds” refers to an association or unionof an antibody or antigen-binding fragment to an antigen with anaffinity or K_(a) (i.e., an equilibrium association constant of aparticular binding interaction with units of 1/M) equal to or greaterthan 10⁵ M⁻¹ (which equals the ratio of the on-rate [K_(on)] to the offrate [K_(off)] for this association reaction), while not significantlyassociating or uniting with any other molecules or components in asample. Alternatively, affinity may be defined as an equilibriumdissociation constant (K_(d)) of a particular binding interaction withunits of M (e.g., 10⁻⁵ M to 10⁻¹³ M). Antibodies may be classified as“high-affinity” antibodies or as “low-affinity” antibodies.“High-affinity” antibodies refer to those antibodies having a K_(a) ofat least 10⁷M⁻¹, at least 10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 10¹⁰ M⁻¹,at least 10¹¹ M⁻¹, at least 10¹²M⁻¹, or at least 10¹³ M⁻¹.“Low-affinity” antibodies refer to those antibodies having a K_(a) of upto 10⁷M⁻¹, up to 10⁶ M⁻¹, up to 10⁵M⁻¹. Alternatively, affinity may bedefined as an equilibrium dissociation constant (K_(d)) of a particularbinding interaction with units of M (e.g., 10⁻⁵ M to 10⁻¹³M).

A variety of assays are known for identifying antibodies of the presentdisclosure that bind a particular target, as well as determining bindingdomain or binding protein affinities, such as Western blot, ELISA,analytical ultracentrifugation, spectroscopy, and surface plasmonresonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y.Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al.,Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, orthe equivalent). Assays for assessing affinity or apparent affinity orrelative affinity are also known. In certain examples, apparent affinityfor an immunoglobulin binding protein is measured by assessing bindingto various concentrations of tetramers, for example, by flow cytometryusing labeled tetramers. In some examples, apparent K_(d) of animmunoglobulin binding protein is measured using 2-fold dilutions oflabeled tetramers at a range of concentrations, followed bydetermination of binding curves by non-linear regression, apparent K_(d)being determined as the concentration of ligand that yieldedhalf-maximal binding.

In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure is capable of reducing motility of the Campylobacterin an in vitro cell motility assay. An exemplary motility assay isillustrated schematically in FIG. 2; see also Riazi et al., PLoS One8(12): e83928 (2013).

In certain embodiments, an antibody of the present disclosure is capableof neutralizing infection by one or more Campylobacter sp. As usedherein, a “neutralizing antibody” is one that can neutralize, i.e.,prevent, inhibit, reduce, impede, or interfere with, the ability of apathogen to initiate and/or perpetuate an infection in a host. The terms“neutralizing antibody” and “an antibody that neutralizes” or“antibodies that neutralize” are used interchangeably herein.

In any of the presently disclosed embodiments, the Campylobactercomprises Campylobacter jejuni, Campylobacter coli, or both. In certainembodiments, the Campylobacter comprises C. jejuni 81-176, C. coli10092/ATB, or both.

Terms understood by those in the art of antibody technology are eachgiven the meaning acquired in the art, unless expressly defineddifferently herein. For example, the term “antibody” refers to an intactantibody comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, as well as anyantigen-binding portion or fragment of an intact antibody that has orretains the ability to bind to the antigen target molecule recognized bythe intact antibody, such as an scFv, Fab, or Fab′2 fragment. Thus, theterm “antibody” herein is used in the broadest sense and includespolyclonal and monoclonal antibodies, including intact antibodies andfunctional (antigen-binding) antibody fragments thereof, includingfragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chainantibody fragments, including single chain variable fragments (scFv),and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. Theterm encompasses genetically engineered and/or otherwise modified formsof immunoglobulins, such as intrabodies, peptibodies, chimericantibodies, fully human antibodies, humanized antibodies, andheteroconjugate antibodies, multispecific, e.g., bispecific antibodies,diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv.Unless otherwise stated, the term “antibody” should be understood toencompass functional antibody fragments thereof. The term alsoencompasses intact or full-length antibodies, including antibodies ofany class or sub-class, including IgG and sub-classes thereof, IgM, IgE,IgA, and IgD.

The terms “V_(L)” or “VL” and “V_(H)” or “VH” refer to the variablebinding region from an antibody light and heavy chain, respectively. Incertain embodiments, a VL is a kappa (κ) class (also “VK” herein). Incertain embodiments, a VL is a lambda (λ) class. The variable bindingregions are made up of discrete, well-defined sub-regions known as“complementarity determining regions” (CDRs) and “framework regions”(FRs). The terms “complementarity determining region,” and “CDR,” aresynonymous with “hypervariable region” or “HVR,” and refer to sequencesof amino acids within antibody variable regions, which, in general,confer the antigen specificity and/or binding affinity of the antibody,and are separated from one another by a framework region. There arethree CDRs in each variable region (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2,LCDR3; also referred to as CDRHs and CDRLs, respectively). In certainembodiments, an antibody VH comprises four FRs and three CDRs asfollows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an antibody VL comprisesfour FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4.In general, the VH and the VL together form the antigen-binding sitethrough their respective CDRs.

As used herein, a “variant” of a CDR refers to a functional variant of aCDR sequence having up to 1-3 amino acid substitutions (e.g.,conservative or non-conservative substitutions), deletions, orcombinations thereof.

Numbering of CDR and framework regions may be according to any knownmethod or scheme, such as the Kabat, Chothia, EU, IMGT, and AHonumbering schemes (see, e.g., Kabat et al., “Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services, PublicHealth Service National Institutes of Health, 1991, 5^(th) ed.; Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987)); Lefranc et al., Dev. Comp.Immunol. 27:55, 2003; Honegger and Plückthun, J. Mol. Mo. 309:657-670(2001)). Equivalent residue positions can be annotated and for differentmolecules to be compared using Antigen receptor Numbering And ReceptorClassification (ANARCI) software tool (2016, Bioinformatics 15:298-300).

In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, andLCDR3 amino acid sequences according to: (i) SEQ ID NOs:9-14,respectively; or (ii) SEQ ID NOs:25-30, respectively.

The term “CL” refers to an “immunoglobulin light chain constant region”or a “light chain constant region,” i.e., a constant region from anantibody light chain. The term “CH” refers to an “immunoglobulin heavychain constant region” or a “heavy chain constant region,” which isfurther divisible, depending on the antibody isotype into CH1, CH2, andCH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM).

A “Fab” (fragment antigen binding) is the part of an antibody that bindsto antigens and includes the variable region and CH1 of the heavy chainlinked to the light chain via an inter-chain disulfide bond. Each Fabfragment is monovalent with respect to antigen binding, i.e., it has asingle antigen-binding site. Pepsin treatment of an antibody yields asingle large F(ab′)2 fragment that roughly corresponds to two disulfidelinked Fab fragments having divalent antigen-binding activity and isstill capable of cross-linking antigen. Both the Fab and F(ab′)2 areexamples of “antigen-binding fragments.” Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CH1 domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of Fab′ fragmentsthat have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This fragment consists ofa dimer of one heavy- and one light-chain variable region domain intight, non-covalent association. From the folding of these two domainsemanate six hypervariable loops (three loops each from the H and Lchain) that contribute the amino acid residues for antigen binding andconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although typically at a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv”, are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

During antibody development, DNA in the germline variable (V), joining(J), and diversity (D) gene loci may be rearranged and insertions and/ordeletions of nucleotides in the coding sequence may occur. Somaticmutations may be encoded by the resultant sequence, and can beidentified by reference to a corresponding known germline sequence. Insome contexts, somatic mutations that are not critical to a desiredproperty of the antibody (e.g., specific binding to a Campylobactersp.), or that confer an undesirable property upon the antibody (e.g., anincreased risk of immunogenicity in a subject administered theantibody), or both, may be replaced by the correspondinggermline-encoded amino acid, or by a different amino acid, so that adesirable property of the antibody is improved or maintained and theundesirable property of the antibody is reduced or abrogated. Thus, insome embodiments, the antibody or antigen-binding fragment of thepresent disclosure comprises at least one more germline-encoded aminoacid in a variable region as compared to a parent antibody or antigenbinding fragment, provided that the parent antibody or antigen bindingfragment comprises one or more somatic mutations. Variable region aminoacid sequences of exemplary anti-Campylobacter antibodies of the presentdisclosure are provided in Table 1 herein, wherein somatic mutations areshown by underlining.

Also provided herein are variant antibodies that comprise one or moreamino acid alterations in a variable region (e.g., VH, VL, framework orCDR) as compared to a presently disclosed (“parent”) antibody, whereinthe variant antibody is capable of specifically binding to aCampylobacter FliD epitope with an affinity similar to or stronger thanthe parent antibody. For example, in some embodiments, an antibody orantigen-binding fragment of the present disclosure comprises a heavychain variable domain (VH) having at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO:2 or 22, and a light chain variabledomain (VL) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acidsequence of SEQ ID NO:4 or 24, provided that the variant antibody orantigen-binding fragment specifically binds a Campylobacter FliD epitopewith an affinity similar to or better than a parent antibody having a VHaccording to SEQ ID NO:2 or 22 and a VL according to SEQ ID NO:4 or 24,respectively.

In certain embodiments, the antibody or antigen-binding fragment cancomprise: (i) VH having at least 85% (i.e., at least 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more) amino acid identity toSEQ ID NO:2, and a VL having at least 85% (i.e., at least 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more) amino acididentity to SEQ ID NO:4; or (ii) VH having at least 85% amino acididentity to SEQ ID NO:22, and a VL having at least 85% amino acididentity to SEQ ID NO:24.

In further embodiments, the antibody or antigen-binding fragmentcomprises: (i) a VH according to SEQ ID NO:2, and a VL according to SEQID NO:4; or (ii) a VH according to SEQ ID NO:22, and a VL according toSEQ ID NO:24.

In any of the presently disclosed embodiments, the antibody orantigen-binding fragment is multispecific; e.g., bispecific,trispecific, or the like.

In any of the presently disclosed embodiments, the antibody orantigen-binding fragment is an IgA, IgG, IgD, IgE, or IgM isotype.

In certain embodiments, the antibody or antigen-binding fragment is anIgA isotype. In humans, IgA antibodies are found in monomeric, dimeric,or tetrameric forms. IgA subclasses include IgA1 and IgA2. IgA1 has alonger hinge sequence (between the Fab arms and the Fc) than IgA2. See,e.g., Woof and Kerr, Immunology 113(2):175-177 (2004)).

Without wishing to be bound by theory, IgA dimers generally comprise twoIgA monomers linked together by at least a joining chain (“J-chain”)polypeptide formed in IgA-secreting cells. Soluble IgA dimers aregenerally capable of forming a complex with poly-Ig receptor (“pIgR”)proteins found on the basolateral surface of epithelial cells. Followingformation, the IgA dimer-pIgR complex is internalized into theepithelial cell and transported to the luminal surface for release intothe lumen. Prior to secretion into the lumen, a portion of the pIgR iscleaved, while a portion known as the secretory component or “SC”remains bound to the IgA, forming secretory IgA (SIgA). Without wishingto be bound by theory, the SC is believed to improve stability of theIgA dimer in the vesicular and luminal environments, possibly byprotecting proteolytically sensitive sites in the IgA dimer.

In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure is an IgA1 isotype or an IgA2 isotype.

In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure comprises an IgA dimer molecule.

In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure comprises a secretory IgA molecule.

The “Fc” fragment or Fc polypeptide comprises the carboxy-terminalportions (i.e., the CH2 and CH3 domains of IgG) of both antibody Hchains held together by disulfides. The effector functions of antibodiesare determined by sequences in the Fc region. The Fc domain is theportion of the antibody recognized by cell receptors, such as the FcRs,and to which the complement-activating protein, Clq, binds. As discussedherein, modifications (e.g., amino acid substitutions) may be made to anFc domain in order to modify (e.g., improve, reduce, or ablate) one ormore functionality of an Fc-containing polypeptide (e.g., an antibody ofthe present disclosure). In any of the presently disclosed embodiments,the antibody or antigen-binding fragment comprises a Fc polypeptide or afragment thereof, including a CH2 (or a fragment thereof, a CH3 (or afragment thereof), or a CH2 and a CH3, wherein the CH2, the CH3, or bothcan be of any isotype and may contain amino acid substitutions or othermodifications as compared to a corresponding wild-type CH2 or CH3,respectively. In certain embodiments, a Fc polypeptide of the presentdisclosure comprises two CH2-CH3 polypeptides that associate to form adimer.

In certain embodiments, the antibody or antigen-binding fragment ofcomprises a heavy chain constant region having at least 90% identity(i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least99% sequence identity to any one of SEQ ID NOs:40-42.

In any of the presently disclosed embodiments, the antibody orantigen-binding fragment is monoclonal. The term “monoclonal antibody”(mAb) as used herein refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present, in some cases in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to polyclonal antibodypreparations that include different antibodies directed againstdifferent epitopes, each monoclonal antibody is directed against asingle epitope of the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they may be synthesizeduncontaminated by other antibodies. The term “monoclonal” is not to beconstrued as requiring production of the antibody by any particularmethod. For example, monoclonal antibodies useful in the presentinvention may be prepared by the hybridoma methodology first describedby Kohler et al., Nature 256:495 (1975), or may be made usingrecombinant DNA methods in bacterial, eukaryotic animal, or plant cells(see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example. Monoclonal antibodies may alsobe obtained using methods disclosed in PCT Publication No. WO2004/076677A2.

Antibodies and antigen-binding fragments of the present disclosureinclude “chimeric antibodies” in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (see, U.S. Pat. Nos.4,816,567; 5,530,101 and 7,498,415; and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)). For example, chimeric antibodiesmay comprise human and non-human residues. Furthermore, chimericantibodies may comprise residues that are not found in the recipientantibody or in the donor antibody. These modifications are made tofurther refine antibody performance. For further details, see Jones etal., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Chimericantibodies also include primatized and humanized antibodies.

A “humanized antibody” is generally considered to be a human antibodythat has one or more amino acid residues introduced into it from asource that is non-human. These non-human amino acid residues aretypically taken from a variable domain. Humanization may be performedfollowing the method of Winter and co-workers (Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)), by substitutingnon-human variable sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. Nos. 4,816,567; 5,530,101 and 7,498,415) whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Insome instances, a “humanized” antibody is one which is produced by anon-human cell or animal and comprises human sequences, e.g., H domains.

A “human antibody” is an antibody containing only sequences that arepresent in an antibody that is produced by a human. However, as usedherein, human antibodies may comprise residues or modifications notfound in a naturally occurring human antibody (e.g., an antibody that isisolated from a human), including those modifications and variantsequences described herein. These are typically made to further refineor enhance antibody performance. In some instances, human antibodies areproduced by transgenic animals. For example, see U.S. Pat. Nos.5,770,429; 6,596,541 and 7,049,426.

In certain embodiments, an antibody or antigen-binding fragment of thepresent disclosure is chimeric, humanized, or human.

Also provided herein are compositions that comprise any antibody orantigen-binding fragment as disclosed herein, and a pharmaceuticallyacceptable carrier, excipient, or diluent. Pharmaceutically acceptablecomponents for use in such compositions are discussed further herein.

In another aspect, the present disclosure provides kits, wherein a kit,comprises: (i) a first antibody or an antigen-binding fragment thereof,which is specific for a Campylobacter flagellum capping protein (FliD)linear epitope; and (ii) a second antibody or an antigen-bindingfragment thereof, which is which is specific for a Campylobacterflagellum capping protein (FliD) conformational epitope.

In certain embodiments, (i) the first antibody or antigen-bindingfragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3sequences according to SEQ ID NOs:9-14, respectively; and (ii) thesecond antibody or antigen-binding fragment comprises HCDR1, HCDR2,HCDR3, LCDR1, LCDR2, and LCDR3 sequences according to SEQ ID NOs:25-30,respectively.

In certain embodiments, (i) the first antibody or antigen-bindingfragment comprises a VH having at least 85% amino acid identity to SEQID NO:2, and a VL having at least 85% amino acid identity to SEQ IDNO:4; and (ii) the second antibody or antigen-binding fragment comprisesa VH having at least 85% amino acid identity to SEQ ID NO:22, and a VLhaving at least 85% amino acid identity to SEQ ID NO:24. In furtherembodiments: (i) the first antibody or antigen-binding fragmentcomprises a VH according to SEQ ID NO:2, and a VL according to SEQ IDNO:4; and (ii) the second antibody or antigen-binding fragment comprisesa VH according to SEQ ID NO:22, and a VL according to SEQ ID NO:24.

In certain embodiments, the first antibody or antigen-binding fragmentand the second antibody or antigen-binding fragment of a kit are each asame isotype. In particular embodiments, the first antibody orantigen-binding fragment and the second antibody or antigen-bindingfragment are each a secreted IgA.

In certain embodiments, a kit further comprises directions orinstructions on using the first and second antibodies or antigen-bindingfragments; e.g., to treat or diagnose a Campylobacter infection in asubject.

Polynucleotides, Vectors, and Host Cells

In another aspect, the present disclosure provides isolatedpolynucleotides that encode any of the presently disclosed antibodies oran antigen-binding fragment thereof. In certain embodiments, thepolynucleotide is codon-optimized for expression in a host cell. Once acoding sequence is known or identified, codon optimization can beperformed using known techniques and tools, e.g., using the GenScript®OptimiumGene™ tool; see also Scholten et al., Clin. Immunol. 119:135,2006). Codon-optimized sequences include sequences that are partiallycodon-optimized (i.e., one or more codon is optimized for expression inthe host cell) and those that are fully codon-optimized.

It will also be appreciated that polynucleotides encoding antibodies andantigen-binding fragments of the present disclosure may possessdifferent nucleotide sequences while still encoding a same antibody orantigen-binding fragment due to, for example, the degeneracy of thegenetic code, splicing, and the like.

In certain embodiments, an isolated polynucleotide encoding aFliD-specific antibody or antigen-binding fragment comprises: (i) aVH-encoding polynucleotide having at least 75% identity (i.e., at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least99% sequence identity) to the nucleotide sequence set forth in any oneof SEQ ID NOs:1, 5, 7, 8, 21, 37, or 38; (ii) a VL-encodingpolynucleotide having at least 75% identity to the nucleotide sequenceset forth in SEQ ID NO:3, 6, 23, or 39; and/or (iii) HCDR1-, HCDR2-,HCDR3-, LCDR1-, LCDR2-, and LCDR3-encoding sequences having at least 90%identity to the nucleotide sequences set forth in SEQ ID NOs:15-20,respectively, or in SEQ ID NOs:31-36, respectively.

Vectors are also provided, wherein the vectors comprise or contain apolynucleotide as disclosed herein (i.e., a polynucleotide that encodesa FliD-specific antibody or antigen-binding fragment). A vector cancomprise any one or more of the vectors disclosed herein.

In a further aspect, the present disclosure also provides a host cellexpressing an antibody or antigen-binding fragment according to thepresent disclosure; or comprising or containing a vector orpolynucleotide according the present disclosure.

Examples of such cells include but are not limited to, eukaryotic cells,e.g., yeast cells, animal cells, insect cells, plant cells; andprokaryotic cells, including E. coli. In some embodiments, the cells aremammalian cells. In certain such embodiments, the cells are a mammaliancell line such as CHO cells (e.g., DHFR-CHO cells (Urlaub et al., PNAS77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells),PER.C6 cells, Y0 cells, Sp2/0 cells. NS0 cells, human liver cells, e.g.Hepa RG cells, myeloma cells or hybridoma cells. Other examples ofmammalian host cell lines include mouse sertoli cells (e.g., TM4 cells);monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidneycells (BHK); African green monkey kidney cells (VERO-76); monkey kidneycells (CV1); human cervical carcinoma cells (HELA); human lung cells(W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalorat liver cells (BRL 3A); mouse mammary tumor (MMT 060562); TM cells;MRC 5 cells; and FS4 cells. Mammalian host cell lines suitable forantibody production also include those described in, for example, Yazakiand Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., HumanaPress, Totowa, N.J.), pp. 255-268 (2003).

In certain embodiments, a host cell is a prokaryotic cell, such as an E.coli. The expression of peptides in prokaryotic cells such as E. coli iswell established (see, e.g., Pluckthun, A. Bio/Technology 9:545-551(1991). For example, antibodies may be produced in bacteria, inparticular when glycosylation and Fc effector function are not needed.For expression of antibody fragments and polypeptides in bacteria, see,e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523.

In particular embodiments, the cell may be transfected with a vectoraccording to the present description with an expression vector. The term“transfection” refers to the introduction of nucleic acid molecules,such as DNA or RNA (e.g. mRNA) molecules, into cells, such as intoeukaryotic cells. In the context of the present description, the term“transfection” encompasses any method known to the skilled person forintroducing nucleic acid molecules into cells, such as into eukaryoticcells, including into mammalian cells. Such methods encompass, forexample, electroporation, lipofection, e.g., based on cationic lipidsand/or liposomes, calcium phosphate precipitation, nanoparticle basedtransfection, virus based transfection, or transfection based oncationic polymers, such as DEAE-dextran or polyethylenimine, etc. Incertain embodiments, the introduction is non-viral.

Moreover, host cells of the present disclosure may be transfected stablyor transiently with a vector according to the present disclosure, e.g.for expressing an antibody, or an antigen-binding fragment thereof,according to the present disclosure. In such embodiments, the cells maybe stably transfected with the vector as described herein.Alternatively, cells may be transiently transfected with a vectoraccording to the present disclosure encoding an antibody orantigen-binding fragment as disclosed herein. In any of the presentlydisclosed embodiments, a polynucleotide may be heterologous to the hostcell.

Accordingly, the present disclosure also provides recombinant host cellsthat heterologously express an antibody or antigen-binding fragment ofthe present disclosure. For example, the cell may be of a species thatis different to the species from which the antibody was fully orpartially obtained (e.g., CHO cells expressing a human antibody or anengineered human antibody). In some embodiments, the cell type of thehost cell does not express the antibody or antigen-binding fragment innature. Moreover, the host cell may impart a post-translationalmodification (PTM; e.g., glysocylation or fucosylation) on the antibodyor antigen-binding fragment that is not present in a native state of theantibody or antigen-binding fragment (or in a native state of a parentantibody from which the antibody or antigen binding fragment wasengineered or derived). Such a PTM may result in a functional difference(e.g., reduced immunogenicity). Accordingly, an antibody orantigen-binding fragment of the present disclosure that is produced by ahost cell as disclosed herein may include one or more post-translationalmodification that is distinct from the antibody (or parent antibody) inits native state (e.g., a human antibody produced by a CHO cell cancomprise a more post-translational modification that is distinct fromthe antibody when isolated from the human and/or produced by the nativehuman B cell or plasma cell).

Insect cells useful expressing a binding protein of the presentdisclosure are known in the art and include, for example, Spodopterafrugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodopterafrugipera SfSWT01 “Mimic™” cells. See, e.g., Palmberger et al., J.Biotechnol. 153(3-4):160-166 (2011). Numerous baculoviral strains havebeen identified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells.

Eukaryotic microbes such as filamentous fungi or yeast are also suitablehosts for cloning or expressing protein-encoding vectors, and includefungi and yeast strains with “humanized” glycosylation pathways,resulting in the production of an antibody with a partially or fullyhuman glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414(2004); Li et al., Nat. Biotech. 24:210-215 (2006).

Plant cells can also be utilized as hosts for expressing a bindingprotein of the present disclosure. For example, PLANTIBODIES™ technology(described in, for example, U.S. Pat. Nos. 5,959,177; 6,040,498;6,420,548; 7,125,978; and 6,417,429) employs transgenic plants toproduce antibodies.

In certain embodiments, the host cell comprises a mammalian cell. Inparticular embodiments, the host cell is a CHO cell, a HEK293 cell, aPER.C6 cell, a Y0 cell, a Sp2/0 cell, a NS0 cell, a human liver cell, amyeloma cell, or a hybridoma cell. In a related aspect, the presentdisclosure provides methods for producing an antibody, antigen bindingfragment, wherein the methods comprise culturing a host cell of thepresent disclosure under conditions and for a time sufficient to producethe antibody, or the antigen-binding fragment. Methods useful forisolating and purifying recombinantly produced antibodies, by way ofexample, may include obtaining supernatants from suitable hostcell/vector systems that secrete the recombinant antibody into culturemedia and then concentrating the media using a commercially availablefilter. Following concentration, the concentrate may be applied to asingle suitable purification matrix or to a series of suitable matrices,such as an affinity matrix or an ion exchange resin. One or more reversephase HPLC steps may be employed to further purify a recombinantpolypeptide. These purification methods may also be employed whenisolating an immunogen from its natural environment. Methods for largescale production of one or more of the isolated/recombinant antibodydescribed herein include batch cell culture, which is monitored andcontrolled to maintain appropriate culture conditions. Purification ofsoluble antibodies may be performed according to methods describedherein and known in the art and that comport with laws and guidelines ofdomestic and foreign regulatory agencies.

Model of Campylobacter Infection

In yet another aspect, the present disclosure provides animal models forinvestigating Campylobacter infection and pathogenesis, as well aspotential therapies and research reagents.

Briefly, existing animal models for studying Campylobacter pathogensishave numerous drawbacks, such as high cost and intensive care settings(e.g., gnotobiotic or germ-free animals), resistance to intestinalcolonization by Campylobacter (e.g., laboratory mice), and unpredictableor deleterious effects of transgenic animals (e.g., SIGIRR or IL10⁻/⁻mice). As described in the Examples, it was found that recently weanedanimals (mice 21 days of age) that are no longer receiving maternalantibodies but do not possess a mature gastrointestinal immune system,and have a depleted intestinal flora, are surprisingly susceptible toinfection by Campylobacter; thus, providing an improved model forstudying Campylobacter pathogenesis and potential treatments thereof.

In certain embodiments, a non-human mammal is provided, wherein thenon-human mammal comprises a weaned mammal that: (i) does not have amature gastrointestinal immune system, and (ii) has a depletedintestinal flora, wherein the depletion is caused by an antibioticagent. In certain embodiments, the non-human mammal further comprises aCampylobacter infection.

In certain embodiments, a non-human mammal of the present disclosure isor comprises a mouse (e.g., a C57BL/6 mouse), a rat, a pig, a rabbit, adog, a cat, a guinea pig, a hamster, a non-human primate (e.g.,cynomolgus), or the like. A non-human mammal that has been weaned is nolonger receiving nutrients via milk from a mother mammal (i.e., themother that gave birth to the non-human mammal, or a surrogate mother).

A mature gastrointestinal immune system according to the presentdisclosure is one that is capable of a functional endogenous immuneactivity (e.g., mucosal protection) against an antigen or pathogen. Forexample, a mature gastrointestinal immune system processes antigens fromvia microfold cells, dendritic cells, and macrophages for presentationto T cells in the gut-associated lymphoid tissue, and producesantigen-neutralizing IgA immunoglobulins by via B cells. See, e.g.,Gutzeit et al., Immunol. Rev. 260(2):76-85 (2014). In certainembodiments, a non-human mammal as disclosed herein does notendogenously produce IgA immunoglobulins, or produces a reduced amountof IgA immunoglobulins as compared to a reference healthy non-humanmammal (i.e., of the same species) that is of an age and/ordevelopmental stage at which the gastrointestinal immune system isconsidered to be mature and functional. A mature gastrointestinal immunesystem typically arises naturally with age in a healthy animal; e.g.,healthy adult mice (56 days) have a mature gastrointestinal immunesystem.

It is understood that commensal bacteria (also referred collectively toas the “flora” or “microbiota”) inhabit the intestine, conferring uponthe host various defensive and metabolic capabilities (see Gutzeit etal., Immunol. Rev. 260(2):76-85 (2014)). The flora may prevent orinhibit colonization by pathogens, such as Campylobacter. A depletedintestinal flora is one that has a statistically significant reductionin one or more of the following: the overall number of bacteria; agrowth rate of one or more of the bacteria; a metabolic function of thebacteria; a defensive function of the bacteria; and/or a diversity ofbacteria, as compared to an intestinal flora of a healthy referencenon-human mammal (i.e., of the same species and the same age, or ofabout the same age).

The age or developmental stage at which such a non-human mammal may beweaned by separation from the mother will be in accordance with therelevant animal care standards and the known biology of the organism.For example, mice may be weaned at about 15 days, about 16 days, about17 days, about 18 days, about 19 days, about 20 days, about 21 days,about 22 days, about 23 days, about 24 days, about 25 days, about 26days, about 27 days, about 28 days, about 29 days, or about 30 daysafter birth, or later. In certain embodiments, a weaned mouse is 18days, 19 days, 20 days, 21 days, 22 days, 23 days, or 24 days old, orolder. In certain embodiments, a non-human mammal is selected at an ageor developmental stage that is less than the age, or is of an earlierdevelopmental stage, respectively, than the age or developmental stageby which the non-human mammal will possess a mature gastrointestinalimmune system. In other embodiments, a non-human mammal may bemanipulated (e.g., genetically or otherwise) to delay or preventdevelopment of a mature gastrointestinal immune system. It will beunderstood that a gastrointestinal immune system may mature over time;accordingly, in preferred embodiments, a non-human mammal is recentlyweaned. A recently weaned mammal is a mammal that has been weaned forfrom about 1 to about 10 days.

A non-human mammal according to the present disclosure has a depletedintestinal flora. Bacteria number, growth rate, metabolic function,defensive function, and diversity can be determined, and compared to areference, using methods known to a person of ordinary skill in the art.

Intestinal flora can be depleted, for example, by administration ofantibiotic agent. Exemplary antibiotic agents include vancomycin andother glycopeptide antibiotics, trimethoprim, ampicillin, metronidazole,and streptomycin, and analogs thereof, and combinations thereof. Inpreferred embodiments, the antibiotic agent is or comprises an agent towhich Campylobacter have resistance; e.g., vancomycin or an analogthereof. Dosing and administration of an antibiotic agent to deplete anintestinal flora can be determined in accordance with known principles,accounting for, e.g., the age, size, and/or health of the non-humanmammal, and the desired effect.

In further embodiments, the non-human mammal further comprises aCamplyobacter (e.g., a Camplyobacter of interest, such as a C. jejuni, aC. coli, or both). Campylobacter can be administered, for example,orally (e.g., via gavage), in an amount sufficient to form colonies inthe intestine. For example, mice aged 12, 21, or 56 days are innoculatedwith 10⁸ to 10⁹ Campylobacter. In certain embodiments, the Campylobacterintroduced to the non-human mammal comprises about 10⁵, about 5×10⁵,about 10⁶, about 5×10⁶, about 10⁷, about 5×10⁷, about 10⁸, about 5×10⁸,about 10⁹, about 5×10⁹, about 10¹⁰, about 5×10⁵, about 10¹¹, about5×10¹¹, or about 10¹² Campylobacter, or more. Once administered, thenumber of Campylobacter may grow to a greater number in the non-humanmammal host.

In a related aspect, methods are provided that comprise administering toor inoculating a weaned non-human mammal that (i) does not have a maturegastrointestinal immune system, and (ii) has a depleted intestinal florawith a Camplyobacter in an amount sufficient to cause an intestinalinfection in the non-human mammal.

In another aspect, methods are provided that comprise administering to anon-human mammal that (i) is weaned, and (ii) does not have a maturegastrointestinal immune system, an agent that depletes an intestinalflora of the non-human mammal. In certain embodiments, the agentcomprises an antibiotic agent as disclosed herein, such as, for example,vancomycin or an analog thereof. In certain embodiments, the methodfurther comprises administering to the non-human mammal, or inoculatingthe non-human mammal with, a Camplyobacter in an amount sufficient tocause an intestinal infection comprising Campylobacter in the non-humanmammal.

Methods and Uses

Also provided herein are methods of treating a subject using an antibodyor antigen-binding fragment of the present disclosure, or a compositioncomprising the same, wherein the subject has, is believed to have, or isat risk for having an infection by a Campylobacter sp. “Treat,”“treatment,” or “ameliorate” refers to medical management of a disease,disorder, or condition of a subject (e.g., a human or non-human mammal,such as a primate, horse, cat, dog, goat, mouse, or rat). In general, anappropriate dose or treatment regimen comprising an antibody orcomposition of the present disclosure is administered in an amountsufficient to elicit a therapeutic or prophylactic benefit. Therapeuticor prophylactic/preventive benefit includes improved clinical outcome;lessening or alleviation of symptoms associated with a disease;decreased occurrence of symptoms; improved quality of life; longerdisease-free status; diminishment of extent of disease, stabilization ofdisease state; delay of disease progression; remission; survival;prolonged survival; or any combination thereof.

A “therapeutically effective amount” or “effective amount” of anantibody, antigen-binding fragment, or composition of this disclosurerefers to an amount of the composition or molecule sufficient to resultin a therapeutic effect, including improved clinical outcome; lesseningor alleviation of symptoms associated with a disease; decreasedoccurrence of symptoms; improved quality of life; longer disease-freestatus; diminishment of extent of disease, stabilization of diseasestate; delay of disease progression; remission; survival; or prolongedsurvival in a statistically significant manner. When referring to anindividual active ingredient, administered alone, a therapeuticallyeffective amount refers to the effects of that ingredient or cellexpressing that ingredient alone. When referring to a combination, atherapeutically effective amount refers to the combined amounts ofactive ingredients or combined adjunctive active ingredient with a cellexpressing an active ingredient that results in a therapeutic effect,whether administered serially, sequentially, or simultaneously. Acombination may comprise, for example, two different antibodies thatspecifically bind a Campylobacter sp. epitope (e.g., a FliD epitope),which in certain embodiments, may be the same or different Campylobactersp., and/or can comprise the same or different epitopes.

Accordingly, in certain embodiments, methods are provided for treating aCampylobacter infection in a subject, wherein the methods compriseadministering to the subject an effective amount of an antibody,antigen-binding fragment, or composition as disclosed herein.

In certain embodiments, methods are provided for reducing (i.e.,reducing or completely abrogating) intestinal inflammation in a subjecthaving a Campylobacter infection, wherein the methods compriseadministering to the subject an effective amount of an antibody,antigen-binding fragment, or composition as disclosed herein.

In certain embodiments, methods are provided for increasing intestinalshedding of a Campylobacter by a subject having a Campylobacterinfection, wherein the methods comprise administering to the subject aneffective amount of an antibody, antigen-binding fragment, orcomposition as disclosed herein.

In any of the presently disclosed embodiments, the antibody orantigen-binding fragment comprises a secretory IgA molecule.

Subjects that can be treated by the present disclosure are, in general,human and other primate subjects, such as monkeys and apes forveterinary medicine purposes. Other model organisms, such as mice andrats, may also be treated according to the present disclosure. In any ofthe aforementioned embodiments, the subject may be a human subject. Thesubjects can be male or female and can be any suitable age, includinginfant, juvenile, adolescent, adult, and geriatric subjects.

Typical routes of administering the presently disclosed compositionsthus include, without limitation, oral, topical, transdermal,inhalation, parenteral, sublingual, buccal, rectal, vaginal, andintranasal. The term “parenteral”, as used herein, includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. In certain embodiments, administering comprisesadministering by a route that is selected from oral, intravenous,parenteral, intragastric, intrapleural, intrapulmonary, intrarectal,intradermal, intraperitoneal, intratumoral, subcutaneous, topical,transdermal, intracisternal, intrathecal, intranasal, and intramuscular.In particular embodiments, a method comprises orally administering theantibody, antigen-binding fragment, or composition to the subject.

Pharmaceutical compositions according to certain embodiments of thepresent invention are formulated so as to allow the active ingredientscontained therein to be bioavailable upon administration of thecomposition to a patient. Compositions that will be administered to asubject or patient may take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of aherein described an antibody or antigen-binding in aerosol form may holda plurality of dosage units. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington: The Science and Practice of Pharmacy, 20thEdition (Philadelphia College of Pharmacy and Science, 2000). Thecomposition to be administered will, in any event, contain an effectiveamount of an antibody or antigen-binding fragment thereof of the presentdisclosure, for treatment of a disease or condition of interest inaccordance with teachings herein.

A composition may be in the form of a solid or liquid. In someembodiments, the carrier(s) are particulate, so that the compositionsare, for example, in tablet or powder form. The carrier(s) may beliquid, with the compositions being, for example, an oral oil,injectable liquid or an aerosol, which is useful in, for example,inhalatory administration. When intended for oral administration, thepharmaceutical composition is preferably in either solid or liquid form,where semi solid, semi liquid, suspension and gel forms are includedwithin the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following may be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gumtragacanth or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent. When thecomposition is in the form of a capsule, for example, a gelatin capsule,it may contain, in addition to materials of the above type, a liquidcarrier such as polyethylene glycol or oil.

The composition may be in the form of a liquid, for example, an elixir,syrup, solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

Liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of the followingadjuvants: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Physiological saline isa preferred adjuvant. An injectable pharmaceutical composition ispreferably sterile.

A liquid composition intended for either parenteral or oraladministration should contain an amount of an antibody orantigen-binding fragment as herein disclosed such that a suitable dosagewill be obtained. Typically, this amount is at least 0.01% of theantibody or antigen-binding fragment in the composition. When intendedfor oral administration, this amount may be varied to be between 0.1 andabout 70% of the weight of the composition. Certain oral pharmaceuticalcompositions contain between about 4% and about 75% of the antibody orantigen-binding fragment. In certain embodiments, pharmaceuticalcompositions and preparations according to the present invention areprepared so that a parenteral dosage unit contains between 0.01 to 10%by weight of antibody or antigen-binding fragment prior to dilution.

The composition may be intended for topical administration, in whichcase the carrier may suitably comprise a solution, emulsion, ointment orgel base. The base, for example, may comprise one or more of thefollowing: petrolatum, lanolin, polyethylene glycols, bee wax, mineraloil, diluents such as water and alcohol, and emulsifiers andstabilizers. Thickening agents may be present in a composition fortopical administration. If intended for transdermal administration, thecomposition may include a transdermal patch or iontophoresis device. Thepharmaceutical composition may be intended for rectal administration, inthe form, for example, of a suppository, which will melt in the rectumand release the drug. The composition for rectal administration maycontain an oleaginous base as a suitable nonirritating excipient. Suchbases include, without limitation, lanolin, cocoa butter andpolyethylene glycol.

A composition may include various materials which modify the physicalform of a solid or liquid dosage unit. For example, the composition mayinclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and may be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients may beencased in a gelatin capsule. The composition in solid or liquid formmay include an agent that binds to the antibody or antigen-bindingfragment of the disclosure and thereby assists in the delivery of thecompound. Suitable agents that may act in this capacity includemonoclonal or polyclonal antibodies, one or more proteins or a liposome.The composition may consist essentially of dosage units that can beadministered as an aerosol. The term aerosol is used to denote a varietyof systems ranging from those of colloidal nature to systems consistingof pressurized packages. Delivery may be by a liquefied or compressedgas or by a suitable pump system that dispenses the active ingredients.Aerosols may be delivered in single phase, bi phasic, or tri phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, and the like, which together may form a kit. One ofordinary skill in the art, without undue experimentation, may determinepreferred aerosols.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. For example, a composition intended tobe administered by injection can be prepared by combining a compositionthat comprises an antibody, antigen-binding fragment thereof, orantibody conjugate as described herein and optionally, one or more ofsalts, buffers and/or stabilizers, with sterile, distilled water so asto form a solution. A surfactant may be added to facilitate theformation of a homogeneous solution or suspension. Surfactants arecompounds that non-covalently interact with the peptide composition soas to facilitate dissolution or homogeneous suspension of the antibodyor antigen-binding fragment thereof in the aqueous delivery system.

In general, an appropriate dose and treatment regimen provide thecomposition(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit (such as described herein, including an improvedclinical outcome (e.g., a decrease in frequency, duration, or severityof diarrhea or associated dehydration, or inflammation, or longerdisease-free and/or overall survival, or a lessening of symptomseverity). For prophylactic use, a dose should be sufficient to prevent,delay the onset of, or diminish the severity of a disease associatedwith disease or disorder. Prophylactic benefit of the compositionsadministered according to the methods described herein can be determinedby performing pre-clinical (including in vitro and in vivo animalstudies) and clinical studies and analyzing data obtained therefrom byappropriate statistical, biological, and clinical methods andtechniques, all of which can readily be practiced by a person skilled inthe art.

Compositions are administered in an effective amount (e.g., to treat aCampylobacter infection), which will vary depending upon a variety offactors including the activity of the specific compound employed; themetabolic stability and length of action of the compound; the age, bodyweight, general health, sex, and diet of the subject; the mode and timeof administration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy. In certain embodiments, following administration oftherapies according to the formulations and methods of this disclosure,test subjects will exhibit about a 10% up to about a 99% reduction inone or more symptoms associated with the disease or disorder beingtreated as compared to placebo-treated or other suitable controlsubjects.

Generally, a therapeutically effective daily dose is (for a 70 kgmammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e.,7.0 g); preferably a therapeutically effective dose is (for a 70 kgmammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e.,3.5 g); more preferably a therapeutically effective dose is (for a 70 kgmammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75g).

In certain embodiments, a method comprises administering the antibody,antigen-binding fragment, or composition to the subject at 2, 3, 4, 5,6, 7, 8, 9, 10 times, or more.

In certain embodiments, a method comprises administering the antibody,antigen-binding fragment, or composition to the subject a plurality oftimes, wherein a second or successive administration is performed atabout 6, about 7, about 8, about 9, about 10, about 11, about 12, about24, about 48, about 74, about 96 hours, or more, following a first orprior administration, respectively.

In certain embodiments, a method comprises administering the antibody,antigen-binding fragment, or composition at least one time prior to thesubject being infected by the Campylobacter.

In any of the presently disclosed methods, following the administering,a stool sample from the subject comprises an increased number ofCampylobacter colony-forming units (CFUs) as compared to a stool samplefrom the subject prior to being administered an effective amount of theantibody, antigen-binding fragment, or composition.

Lipocalin-2 (LCN2) is a marker of intestinal inflammation and is linkedto epithelial damage and neutrophil infiltration. In any of thepresently disclosed methods, following the administering, a stool samplefrom the subject comprises a reduced amount of LCN2 as compared to astool sample from the subject prior to being administered an effectiveamount of the antibody, antigen-binding fragment, or composition. LCN2can be measured, for example, using anti-LCN2 antibody and performing anELISA assay.

In any of the presently disclosed methods, following the administering,the subject comprises a reduced amount of polymorphonucleated (PMN) cellinfiltrate in the subject's caecum as compared to the subject prior tobeing administered an effective amount of the antibody, antigen-bindingfragment, or composition, wherein the PMN cells are Gr1⁺CD11b⁺.

In any of the presently disclosed methods, following the administering,the subject has an improved caecum histology as compared to the subjectprior to being administered an effective amount of the antibody,antigen-binding fragment, or composition. Standard histology analysisand scoring techniques may be employed to score a tissue (e.g., caecum)for damage, inflammation, or other indicia of a Campylobacter infection.

In any of the presently disclosed methods, following the administering,the antibody or antigen-binding fragment is present in the caecum and/orin feces of the subject for at least 4 hours or for at least 8 hoursfollowing the administration.

Compositions comprising an antibody or antigen-binding fragment of thepresent disclosure may also be administered simultaneously with, priorto, or after administration of one or more other therapeutic agents.Such combination therapy may include administration of a singlepharmaceutical dosage formulation which contains a compound of theinvention and one or more additional active agents, as well asadministration of compositions comprising an antibody or antigen-bindingfragment of the disclosure and each active agent in its own separatedosage formulation. For example, an antibody or antigen-binding fragmentthereof as described herein and the other active agent can beadministered to the patient together in a single oral dosage compositionsuch as a tablet or capsule, or each agent administered in separate oraldosage formulations. Similarly, an antibody or antigen-binding fragmentas described herein and the other active agent can be administered tothe subject together in a single parenteral dosage composition such asin a saline solution or other physiologically acceptable solution, oreach agent administered in separate parenteral dosage formulations.Where separate dosage formulations are used, the compositions comprisingan antibody or antigen-binding fragment and one or more additionalactive agents can be administered at essentially the same time, i.e.,concurrently, or at separately staggered times, i.e., sequentially andin any order; combination therapy is understood to include all theseregimens.

In a related aspect, uses of the presently disclosed antibodies,antigen-binding fragments, and compositions are provided.

In certain embodiments, an antibody, antigen-binding fragment, orcomposition is provided for use in a method of: (a) treating aCampylobacter infection in a subject; (b) reducing intestinalinflammation in a subject having a Campylobacter infection; and/or (c)increasing intestinal shedding of a Campylobacter by a subject having aCampylobacter infection. It will be appreciated that treatment,reduction of inflammation, and increased intestinal shedding are asdescribed herein.

In certain embodiments, an antibody, antigen-binding fragment, orcomposition is provided for use in a method of manufacturing orpreparing a medicament for: (a) treating a Campylobacter infection in asubject; (b) reducing intestinal inflammation in a subject having aCampylobacter infection; and/or (c) increasing intestinal shedding of aCampylobacter by a subject having a Campylobacter infection. In certainembodiments, the medicament is formulated for oral administration.

EXAMPLES Example 1 Experimental Methods

Immunoglobulins against select Campylobacter antigens associated withbacterial motility, adhesion, or mucosa invasion were isolated andtested for potency, selectivity, and breadth in vitro and ex-vivo. Thecorresponding recombinant SIgA (rSIgA) were expressed viaco-transfection in mammalian cells and purified using affinity columnchromatography.

rSIgA ability to curb Campylobacter motility was appraised in vitro bymotility assay (Riazi et al., PLoS One 2013), whereas breadth andcross-reactivity of the rSIgA with the murine microbiota was evaluatedby incubating the mAbs with the stools of infected or mock infected micefollowed by FACS analysis of human IgA coated bacteria.

The prophylactic activity of orally administered rSIgA was tested inC57BL/6 mouse model for Campylobacter infection. In a first experiment,C57/BL6 mice were pre-treated 3 times via oral gavage with vancomycin 48hours before mAbs administration. Next, after 1 hour the animals wereinfected with 10⁹ CFU of Campylobacter jejuni strain 81-176 (collectionnumber ATCC BAA-2151) and then administered again twice with theantibodies at 6-hour intervals. In another experiment, C57BL/6 femalemice (21 days old) were pre-treated with 10 mg of vancomycin(Sigma-Aldrich) in 200 μl PBS 48, 24 and 12 h prior to mAbsadministration. Mice then received a single oral administration of 200μg of FliD-reactive mAbs in 200 μl PBS 2 hours before being infected byoral gavage with 10⁸ CFU of C. jejuni 81-176 (collection number ATCCBAA-2151). This infection can also be done with other Campylobacterspecies, such as Campylobacter coli strain 10092/ATB (collection numberNCTC 11437). Bacterial shedding in animal stools was monitoredthroughout the experiment. Lipocalin-2, a marker of intestinalinflammation, was measured by ELISA in stool samples and histologicalevaluation on the caecum was performed to investigate bacterial invasionand changes of the mucosal epithelium.

Example 2 Generation of a Campylobacter-Specific Monoclonal IgAAntibodies

Campylobacter flagellar capping protein (FliD) has not been assessedto-date as a potential target for therapeutic monoclonal antibodies.FliD was selected as antigen for mAb development. The frequencies ofIgG+ and IgA+FliD-reactive memory B cells in 50 tonsillar samples ofSwiss origin were evaluated using theAntigen-specific-Memory-B-cell-Repertoire-Analysis (AMBRA) (Pinna etal., Eur. J. Immunol., 39:1260-1270 (2009)) (FIG. 13). IgG+ and IgA+memory B cells from selected tonsils were then immortalized (Traggiai etal., Nat. Med., 10:871-875 (2004)), and culture supernatants screenedusing a 384 well plate-based high-throughput platform to identify cellclones expressing FliD-reactive antibodies.

Memory B cell clones producing human monoclonal antibodies “CAA” and“CCG4” were isolated and selected based on their specificity andaffinity for FliD antigen. CAA1 was isolated as an IgA1 encoded byV_(H)3-48/D2-15/J_(H)3 with a 21-amino acid HCDR3 and V_(K)1-39/J_(K)S.CCG4 was isolated as an IgG3 encoded by V_(H)3-9/D1-7/J_(H)1 bearing ashorter (11 amino acids) HCDR3 and V_(L)3-27/J_(L)3. Nucleic acid andamino acid sequences of variable regions from exemplary mAbs areprovided in Table 1.

Humans present two IgA isotype subclasses that differ mainly in thelength and glycosylation of the hinge region. IgA1 possesses a hingethat is 13 amino acids longer than that of IgA2 and contains up to fiveO-linked glycans at serine and threonine residues. The longer hinge ofIgA1 is believed to confer greater flexibility and a longer Fab reach,but may also contribute to sensitivity of IgA1 to IgA1 proteases. IgA2has a shorter hinge region that lacks proline-serine and/orproline-threonine peptide bonds, and is resistant to IgA1 proteases(Plaut, Annu. Rev. Microbiol., 37:603-622 (1983)). In addition, the IgA2isotype can undergo reverse transcytosis by contacting Dectin-I receptoron the surface of PPs M cells (Rochereau et al., PLoS Biol., 11:e1001658(2013)). IgG, IgM, and IgA1 isotypes are not believed to have thisability. Both the Cal region and the glycosylation pattern of IgA2 arethought to be important for interaction with Dectin-I receptor, whichmay boost adaptive immunity against pathogens (Rochereau et al., Eur. J.Immunol., 45:773-779 (2015)).

The IgA2 scaffold was initially selected over IgA1 for further studies,and both CAA1 and CCG4 were produced as rSIgA2 before further in vitrocharacterization. For a control antibody non-reactive with the antigen,HGN194 mAb (Corti et al., PLoS One, 5:e8805 (2010)), which targets anHIV glycoprotein, was also expressed as rSIgA2. Antibodies wereexpressed as rSIgA2 via plasmid co-transfection in mammalian cells andpurified using CaptureSelect IgA affinity columns.

Structural and functional characterization of purified rSIgA wasperformed using ELISA and UPLC analysis. Campylobacter-reactive rSIgAwas able to recognize and bind the most common Campylobacter speciesassociated with severe infections, including recent clinical isolates(FIG. 1), displaying limited cross-reactivity with the murinemicrobiota, which might be due to modest level of homology with similarantigens in the flagella of commensal species or to non-Fab mediatedinteractions.

Table 1 provides sequences of exemplary anti-FliD mAbs according to thepresent disclosure, as well as sequences of exemplary FliD proteins.Antibody CDR sequences (amino acid and nucleotide) are shown in bold.Antibody residues that arose from somatic mutation are underlined.

TABLE 1 Sequences Sequence Description SEQ ID NO. Sequence CAA1 1gaagcacagctggtggagagcggcggcggcctgatcc (VH, codon optimizedagccaggcggctctctgagactgagctgtgaggcctctg for IgA2m2)gcttcagcctgagctcccacgagatgaactgggtgagacaggcacctggcaagggactggagtggctgagctacatctccacctctggcatcacaatctactatgcagactccgtgcggggccggttcaccatcagcagggatacagccaagaactccctgtacctgcagatgaattctctgagggccgaggacaccgccctgtatcactgtgcccgcgatctgggcggctactgctctggcggcctgtgctatcctcgcggcgccctggacctgtggggacagggaaccacagtgaccgtgtctagcg CAA1 2 EAQLVESGGGLIQPGGSLRLSCEASG (VH)F SL SS H EMNWVRQAPGKGLEWLSYI S T SG I TIYYADSVRGRFTISRDTAKNSLYLQMNSLRAEDTALYHCARDLGG YCSGG L CYPRGA L D L WGQGTTVTV SS CAA1 3gacatcctgatgacacagtctcctagctccctgtctgcctct (VK, codongtgggcgatagggtgaccatcacatgccgcgcctcccag optimized)acaatccggacctacgtgaactggtatcagcagaagcccggcgagacacctaggctgctgatctacgcagcaaccatcctgcagcggggcgtgccatccagattctccggctctggcagcggcacagactttaccctgacaatcacctctctgcagcccgaggatttcggcacctactattgtcagcagaattataagacattcctgacctttggccagggcacccggctggagatca agc CAA1 4DILMTQSPSSLSASVGDRVTITCRASQ (VK) TIRTYVNWYQQKPGETPRLLIYAATILQRGVPSRFSGSGSGTDFTLTITSLQP EDFGTYYCQQNYKTFLTFGQGTRLE IK CAA1 5GAGGCGCAGCTGGTGGAGTCTGGG (VH, native) GGAGGCCTGATACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGAAGCCT CTGGCTTCTCCCTCAGTTCTCATGAAATGAATTGGGTCCGCCAGGCTC CAGGGAAGGGGCTGGAGTGGCTTTCATATATTAGTACTAGTGGTATTA CAATATATTACGCGGACTCTGTGAGGGGCCGATTCACCATCTCCAGAG ACACCGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGA GGACACGGCTCTTTATCACTGTGCGAGAGATCTTGGCGGTTATTGTAG TGGTGGTTTGTGCTACCCGAGGG GTGCCTTGGATCTCTGGGGCCAAGGGACAACGGTCACCGTCTCGTCAG CAA1 6 GACATCCTGATGACCCAGTCTCCAT (VK, native)CCTCCCTGTCTGCATCTGTCGGAGA CAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTCGCACCTATGT AAATTGGTATCAGCAGAAGCCAGGGGAAACCCCAAGACTCCTTATCTAT GCTGCAACCATTTTGCAGAGAGGGGTCCCATCAAGGTTCAGTGGCAGTG GATCTGGGACAGATTTCACTCTCACCATTACCAGTCTGCAACCTGAAGAT TTTGGAACTTACTACTGTCAACAGAACTACAAAACCTTTCTCACCTTCG GCCAAGGGACACGACTGGAGATTA AAG CAA1 7GAAGCACAGCTGGTGGAGAGCGGC (VH, codon optimized GGCGGCCTGATCCAGCCAGGCGGCfor IgA1 backbone) TCTCTGAGACTGAGCTGTGAGGCAT CTGGCTTCAGCCTGAGCTCCCACGAGATGAACTGGGTGAGACAGGCACC TGGCAAGGGCCTGGAGTGGCTGAGCTACATCTCCACCTCTGGCATCACA ATCTACTATGCAGACTCCGTGCGGGGCCGGTTCACCATCAGCAGGGATA CAGCCAAGAACTCCCTGTACCTGCAGATGAATTCTCTGAGGGCCGAGGA CACCGCCCTGTATCACTGTGCCCGCGATCTGGGCGGCTACTGCAGCGGC GGCCTGTGCTATCCTCGCGGCGCCCTGGACCTGTGGGGACAGGGAACCA CAGTGACCGTGTCTAGCGCCTCCCCAACATCTCCCAAGGTGTTCCCCCTG AGCCTGTGCTCCACACAGCCTGATGGCAACGTGGTCATCGCCTGTCTGGT GCAGGGCTTCTTTCCTCAGGAGCCACTGTCTGTGACATGGTCTGAGTCTG GACAGGGAGTGACAGCACGGAATTTTCCCCCTTCCCAGGACGCCTCTGG CGATCTGTAT CAA1 8 GAGGCCCAGCTGGTGGAAAGCGGC(VH codon optimized GGCGGCCTGATTCAGCCCGGCGGC for IgG1 backbone)TCTCTGAGACTGAGCTGTGAGGCAT CTGGCTTCTCCCTGAGCTCCCACGAGATGAACTGGGTGAGACAGGCACC TGGCAAGGGCCTGGAGTGGCTGTCCTACATCTCCACCTCTGGCATCACA ATCTACTATGCCGACTCTGTGCGGGGCCGGTTCACCATCTCCAGGGATAC AGCCAAGAACTCTCTGTACCTGCAGATGAATAGCCTGAGGGCCGAGGAC ACCGCCCTGTATCACTGTGCACGCGATCTGGGCGGCTACTGCAGCGGCG GCCTGTGCTATCCAAGAGGCGCCCTGGACCTGTGGGGACAGGGAACCAC AGTGACAGTGTCTAGC CAA1 9 GF SL SS H E (HCDR1)CAA1 10 IS T SG I TI (HCDR2) CAA1 11 ARDLGGYCSGG L CYPRGA L D L (HCDR3)CAA1 12 Q T I RT Y (LCDR1) CAA1 13 AA T (LCDR2) CAA1 14 QQ N Y K T F LT(LCDR3) CAA1 15 GGCTTCTCCCTCAGTTCTCATGAA (HCDR1; native) CAA1 16ATTAGTACTAGTGGTATTACAATA (HCDR2; native) CAA1 17GCGAGAGATCTTGGCGGTTATTGTA (HCDR3; native) GTGGTGGTTTGTGCTACCCGAGGGGTGCCTTGGATCTC CAA1 18 CAGACCATTCGCACCTAT (LCDR1; native) CAA1 19GCTGCAACC (LCDR2; native) CAA1 20 CAACAGAACTACAAAACCTTTCTCA(LCDR3; native) CC CCG4 21 GAAGTGCAGCTGGTGGAGTCTGGG (VH, native)GGAGGCTTGGTACAGCCTGGCAGG TCCCTGAGACTCTCCTGCGCAGCCTCTGGAATCACCTTTGATGAATATG CCATGTACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCT CAGGTATTAGTTGGAACAGTGCTAATATAGGCTATGCGGACTCTGTG AAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTCTAT CTGCAAATGAATAGTCTGAGAGCTGAAGACACGGCCTTGTATTACTGTT CAGGTATAACTGGGACTACGGGGATACAGTACTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCAG CCG4 22EVQLVESGGGLVQPGRSLRLSCAAS (VH) G I TFD E YAMYWVRQAPGKGLEWVS GISWNS ANIGYADSVKGRFTISRDNA KKSLYLQMNSLRAEDTALYYC S GIT GTTGIQ Y WGQGTLVTVSS CCG423 TCCTATGAGCTGACACAGCCATCCT (VL, native) CAGTGTCAGTGTCTCCGGGACAGACAGCCAGGATCACCTGCTCAGGAG ATGTATTGGCAAATACATATGCTCGGTGGTTCCAGCAGAAGCCAGGCC AGGCCCCTGTACTGGTGATTTATAAAGACAGTGAGCGGCCCTCAGGGAT CCCTGAGCGATTCTCCGGCTCCAGCTCAGGGACCACAGTCACCTTGATCA TCAGGGGGGCCCAGGTTGAGGATGAGGCTGACTATTACTGTTACTCTG CGGCTGACAACAATCGGAGGGTGTTCGGCGGAGGGACCAAGCTGACC GTCCTAG CCG4 24 SYELTQPSSVSVSPGQTARITCSGDVL(VL) A NT YARWFQQKPGQAPVLVIYKDSE RPSGIPERFSGSSSGTTVTLIIRGAQVEDEADYYCYSAADNNRRVFGGGTKL TVL CCG4 25 G I TFD E YA (HCDR1) CCG4 26 ISWNSAN I (HCDR2) CCG4 27 S GITGTTGIQ Y (HCDR3) CCG4 28 VLA NT Y (LCDR1) CCG429 KDS (LCDR2) CCG4 30 YSAADNNRRV (LCDR3) CCG4 31GGAATCACCTTTGATGAATATGC (HCDR1; native) C CCG4 32ATTAGTTGGAACAGTGCTAATAT (HCDR2; native) A CCG4 33TCAGGTATAACTGGGACTACGGG (HCDR3; native) GATACAGTAC CCG4 34GTATTGGCAAATACATAT (LCDR1; native) CCG4 35 AAAGACAGT (LCDR2; native)CCG4 36 TACTCTGCGGCTGACAACAATCG (LCDR3; native) GAGGGTG CCG4 37gaggtgcagctggtggaaagcggcggcggcctggtgc (VH, codon optimizedagccaggccggtctctgagactgtcttgtgcagcatctgg for IgA2M2aatcaccttcgacgagtatgcaatgtattgggtgcggcag backbone)gcaccaggcaagggactggagtgggtgtccggcatctcttggaacagcgccaatatcggctacgccgactccgtgaagggcaggtttacaatctcccgcgataacgccaagaagtctctgtatctgcagatgaatagcctgagggccgaggataccgccctgtactattgctctggcatcacaggcaccacaggcatccagtactggggccagggcaccctggtgacagtgagctccgcctccccaacctctcccaaggtgttccccctgagcctggactccacacctcaggatggcaacgtggtggtggcctgtctggtgcagggcttctttcctcaggagccactgagcgtgacctggtctgagagcggccagaacgtgacagcccggaattttcccccttctcaggacgccagcggcgatctgtatacc CCG4 38gaggtgcagctggtggaaagcggcggcggcctggtgc (VH, codon optimizedagcctggccggagcctgagactgtcttgtgcagcatctgg for IgG1 backbone)aatcaccttcgacgagtacgccatgtattgggtgcggcaggcacctggcaagggcctggagtgggtgtctggcatcagctggaactccgccaatatcggctacgccgactctgtgaagggcaggtttacaatctctcgcgataacgccaagaagagcctgtatctgcagatgaattccctgagggccgaggataccgccctgtactattgtagcggcatcacaggcaccacaggcatccagtactggggccagggcaccctggtgacagtgagctc c CCG4 39agctacgagctgacccagcctagctccgtgtctgtgagcc (VL, codon optimized)ctggacagacagcaagaatcacatgctctggcgacgtgctggccaacacatacgccaggtggtttcagcagaagcctggacaggcccccgtgctggtcatctacaaggattccgagaggccatctggcattcctgagcggttcagcggctctagctccggcaccacagtgaccctgatcattagaggcgcccaggtggaggatgaggcagattactattgttatagcgccgccgacaacaatcggagagtgttcggcggcggaaccaagctgac agtgctg IgA1-heavy chain 40asptspkvfplslcstqpdgnvviaclvqgffpqeplsvt constant regionwsesgqgvtarnfppsqdasgdlyttssqltlpatqclagksvtchvkhytnpsqdvtvpcpvpstpptpspstpptpspscchprlslhrpaledlllgseanltctltglrdasgvtftwtpssgksavqgpperdlcgcysyssvlpgcaepwnhgktftctaaypesktpltatlsksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrlagkpthvnvsvvmaevdgtcy IgA2(m1)-heavy 41asptspkvfplsldstpqdgnvvvaclvqgffpqeplsv chain constant regiontwsesgqnvtarnfppsqdasgdlyttssqltlpatqcpdgksvtchvkhytnpsqdvtvpcpvpppppcchprlslhrpaledlllgseanltctltglrdasgatftwtpssgksavqgpperdlcgcysyssvlpgcaqpwnhgetftctaahpelktpltanitksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttfavtsilrvaaedwkkgdtfscmvghealplaftqktidrl agkpthvnvsvvmaevdgtcyIgA2(m2)-heavy 42 asptspkvfplsldstpqdgnvvvaclvqgffpqeplsvchain constant region twsesgqnvtarnfppsqdasgdlyttssqltlpatqcpdgksvtchvkhytnssqdvtvpervpppppcchprlslhrpaledlllgseanltctltglrdasgatftwtpssgksavqgpperdlcgcysvssvlpgcaqpwnhgetftctaahpelktpltanitksgntfrpevhllpppseelalnelvtltclargfspkdvlvrwlqgsqelprekyltwasrqepsqgtttyavtsilrvaaedwkkgetfscmvghealplaftqktidr magkpthinvsvvmaeadgtcyCampylobacter jejuni 43 mafgslsslg fgsgvltqdt idklkeaeqksubsp. jejuni serotype aridpytkki eenttkqkdl teiktkllsf qtavssladaO:23/36 (strain 81- tvfakrkvvg sisdnppasl tvnsgvalqs176) Flagellar hook- mninvtqlaq kdvyqskgla ndsgfinanlassociated protein 2 tgttdltffs ngkeytvtvd knttyrelad kineasggei(Uniprot vakivntgek gtpyrltlts ketgedsais A0A0H3PIU8)fyagkkdsng kytsdseaet ifknlgweld ttssidpakd kkgygikdas lhiqtagnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdf egvtkamqdl vdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsm qdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev ikqgslnqyl dssgtgnkgl dfkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqn lanhinskgi eglkvkvesy dqngvkgfkl nfsgdgssdf sikgnatilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltnniks lntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn Campylobacter jejuni 44mafgslsslg fgsgvltqdt idklkeaeqk subsp. jejuni serotypearidpytkki eenttkqkdl teiktkllsf qtavsslada O:2 (strain ATCCtvfakrkvvg sisdnppasl tvnsgvalqs 700819/NCTCmninvtqlaq kdvyqskgla ndggfvnaql 11168) (Uniprotngtadltffs ngkeytvtvd knttyrdlad Q9PHW6)kineasggei vakivntgek gtpyrltlts ketgedsais fyagkkdsng kyqkdinaekifddlgwgld vsasidpdkd kkgygikdas lhiqtaqnae ftldgikmfr ssntvtdlgvgmtltlnktg einfdvqqdf egvtkamqdl vdayndlvtn lnaatdynse tgtkgtlqgisevnsirssi ladlfdsqvv dgttedangn kvntkvmlsm qdfglslnda gtlsfdsskfeqkvkedpds tesffsnitk yedinhtgev iktgslskyl nsnggntngl efkpgdftivfnnqtydlsk nsdgtnfklt gkteeellqn lanhinskgi eglkvkvesy nqnnvtgfrlnfsgdgssdf sikgdanilk elglsdvnit skpiegkgif sklkatlqem tgkdgsitkydesltndiks lntskdstqa midtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar hook- 45 mafgslsslg fgsgvltqdt idklkeaeqk associated protein 2aridpytkki eenttkqkdl teiktkllsf qtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni NCTCmninvtqlaq kdvyqskgla ndggfvnaql 11168 = ATCCngtadltffs ngkeytvtvd knttyrdlad 700819]kineasggei vakivntgek gtpyrltlts NCB Referenceketgedsais fyagkkdsng kyqkdinaek Sequence:ifddlgwgld vsasidpdkd kkgygikdas YP_002343979.1lhiqtaqnae ftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdf egvtkamqdlvdayndlvtn lnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvv dgttedangnkvntkvmlsm qdfglslnda gtlsfdsskf eqkvkedpds tesffsnitk yedinhtgeviktgslskyl nsnggntngl efkpgdftiv fnnqtydlsk nsdgtnfklt gkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdf sikgdanilk elglsdvnitskpiegkgif sklkatlqem tgkdgsitky desltndiks lntskdstqa midtrydtmanqwlqyesil nklnqqlntv tnminaanns nn flagellar filament 46mafgslsslg fgsgvltqdt idklkeaeqk capping protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni] mninvtqlaq kdvyqsqglandsgfinanlNCBI Reference agttdltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_038400380.1fyagkkdaqg qyksdleaek ifkslgweld ttssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl afkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdgsilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn flagellar hook-47 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejunimninvtqlaq kdvyqskglandsgfinanl 81-176]tgttdltffs ngkeytvtvd knttyrelad kineasggei GenBank:vakivntgekgtpyrltlts ketgedsais EAQ73028.1fyagkkdsng kytsdseaet ifloilgweld ttssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev ikqgslnqyldssgtgnkgl dfkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqnlanhinskgieglkvkvesy dqngvkgfkl nfsgdgssdfsikgnatilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltnnikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 48 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqskglandggfvnaql NCBI Referencengtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_010790846.1fyagkkdsng kyqkdtnaek ifddlgwgld asasidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlnfdsskf eqkvkedpdsaesffsnitkyedinhtgei iktgslskyl nsnggntngl dfkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy dqnnvkgfkl nfsgdgssdfsikgdasilkelglpdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn MULTISPECIES:49 mafgslaslg fgsgvltqdt idklkeaeqk flagellar filamentaridpytkki eenttkqkdl teiktkllsfqtavsslada capping protein FliDtvfakrkvvg sisdnppasl tvnsgvalqs [Campylobacter]mninvtqlaq kdvyqsqglandggfvnanl NCBI Referencengtadltffs ngkeytvtvd rnttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_004316510.1fyagkkdang aykndpnaet ifknlgweld atssidlakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdginfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlstv tnminaanns nnflagellar filament 50 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqsqglandggfvnaql NCBI Referencengtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_004306838.1fyagkkdaqg qyksdleaek ifkslgweld ttssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf ekkvkedpds aesffsnitkyedinhtgev iktgslskylnsnggsangl dfkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqnlanhinskgieglkvkvesy dqnnvkgfkl nfsgdgssdf sikgdanilkelglsdvnis skpiegkgifsklkatlqem tgkdgsitky desltndiks lntskdstqvmidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn flagellar filament 51mafgslsslg fgsgvltqdt idklkeaeqk capping protein FliDaridpytkki eenttkqkdl teiktkllsf qtavsslada [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni] mninvtqlaq kdvyqskglandsgfvnaqlNCBI Reference ngtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002935293.1fyagkkdsng kyqkdtnaek ifddlgweld vsasidpdkdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgyftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 52 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqskglandggfvnaql NCBI Referencengtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002928464.1fyagkkdsng kyqkdtnaek ifddlgwgld asasidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlnfdsskf eqkvkedpdsaesffsnitkyedinhtgei iktgslskyl nsnggntngl dfkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy dqnnvkgfkl nfsgdgssdfsikgdasilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 53 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqsqglandggfvnakl NCBI Referencengtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002924910.1fyagkkdvqg qyksdseaek ifkslgweld ttssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 54 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqskglandsgfinanl NCBI Referencetgttdltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekdtpyrltlts ketgedsais WP_002921586.1fyagkkdsng kytsdseaet ifknlgweld ttssidpakd kkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev ikqgslnqyldssgtgnkgl dfkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqnlanhinskgieglkvkvesy dqngvkgfkl nfsgdgssdfsikgnatilqelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 55 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqskglandggfvnaql NCBI Referencengtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002908989.1fyagkkdsng kyqkdtnaek ifddlgwgld vsasidpdkdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev ikqgslnqyldssgtgnkgl dfkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqnlanhinskgieglkvkvesy nqnnvtgfkl nfsgdgssdf sikgnasilkelglsdvnit skpiegkgifsklkatlqem tgkdgsitky desltndiks lntskdstqamidtrydtma nqwlqyesilnklnqqlntv tnminaanns nn flagellar filament 56mafgslsslg fgsgvltqdt idklkeaeqk capping protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni] mninvtqlaq kdvyqskglandsgfinanlNCBI Reference tgttdltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002901368.1fyagkkdsng kytsdleakt ifknlgweld ttssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev ikqgslnqyldssgtgnkgl dfkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqnlanhinskgieglkvkvesy nqnnvtgfrl nfsgdgssdf sikgdanilkelglsdvnit skpiegkgifsklkatlqem tgkdgsitky desltndiks lntskdstqamidtrydtma nqwlqyesilnklnqqlntv tnminaanns nn MULTISPECIES: 57mafgslsslg fgsgvltqdt idklkeaeqk flagellar filamentaridpytkki eenttkqkdl teiktkllsfqtavsslada capping protein FliDtvfakrkvvg sisdnppasl tvnsgvalqs [Campylobacter]mninvtqlaq kdvyqskglandggfinanl NCBI Referencetgttdltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002892358.1fyagkkdsng aykndpnaet ifknlgweld tttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpd stesffsnitkyedinhtge vikqgslnqyldssgtgnkg ldfkpgdfti vfnnqtydls knsdgtnfkltgkteeellq nlanhinskgieglkvkves ydqngvkgfk lnfsgdgssdfsikgnatilqelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar filament 58 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqsqglandggfvnakl NCBI Referencengtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002874097.1fyagkkdaqg qyksdseaek ifkslgweld ttssidpakdkkgysikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 59 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs jejuni]mninvtqlaq kdvyqskglandggfvnaql NCBI Referencengtadltffs ngkeytvtvd knttyrdlad Sequence:kineasggei vakivntgekgtpyrltlts ketgedsais WP_002873395.1fyagkkdsng kyqkdtnaek ifddlgwgld vsasidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn flagellar hook-60 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejunimninvtqlaq kdvyqskglandggfvnaql 305] ngtadltffs ngkeytvtvd knttyrdladGenBank: kineasggei vakivntgekgtpyrltlts ketgedsais EFV08769.1fyagkkdsng kyqkdinaek ifddlgwgld vsasidpdkdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn flagellar hook-61 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejunimninvtqlaq kdvyqskglandsgfvnanl 327] tgttdltffs ngkeytvtvd knttyrdladGenBank: kineasggei vakivntgekgtpyrltlts ketgedsais EFV10698.1fyagkkdaqg qyqsdpeaen ifsnlgweld kttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpd stesffsnitkyedinhtge vikqgslnqyldssgtgnkg ldfkpgdfti vfnnqtydls knsdgtnfkltgkteeellq nlanhinskgieglkvkves ydqngvkgfk lnfsgdgssdfsikgnatilqelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar hook- 62 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejunimninvtqlaq kdvyqsqglandsgfinanl 84-25] agttdltffs ngkeytvtvd knttyrdladkineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyqsdpeaek ifsnlgweld kttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpd stesffsnitkyedinhtge viktgslskylnsnggntng lefkpgdfti vfnnqtydls knsdgtnfkltgkteeellq nlanhinskgieglkvkves ynqnnvtgfr lnfsgdgssdfsikgdanilkelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar hook- 63 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejunimninvtqlaq kdvyqskglandsgfinanl HB93-13]tgttdltffs ngkeytvtvd ksttyrdlad kineasggei GenBank:vakivntgekgtpyrltlts ketgedsais EAQ60315.1fyagkkdaqgqyksdseaekifsnlgweldkttqtidpakdkkgygikda slhiqtaqna eftldgikmf rssntvtdlg vgmtltlnktgeinfdvqqdfegvtkamqd lvdayndlvt nlnaatdyns etgtkgtlqg isevnsirssiladlfdsqvvdgttedang nkvntkvmls mqdfglslnd agtlsfdssk feqkvkedpdstesffsnitkyedinhtge vikqgslnqy ldssgtgnkg lefkpggfti vfnnqtydlsknsdgtnfkltgkteeellq nlanhinskg ieglkvkves ydqngvkgfk lnfsgdgssdfsikgdanilkelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar hook- 64 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejunimninvtqlaq kdvyqskglandggfvnaql 260.94] ngtadltffs ngkeytvtvd knttyrdladGenBank: kineasggei vakivntgekgtpyrltlts ketgedsais EAQ58732.1fyagkkdsng kyqkdtnaek ifddlgwgld asasidpakdkkgygikdas lhiqtagnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlnfdsskf eqkvkedpdsaesffsnitkyedinhtgei iktgnlskyl nsnggntngl dfkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy dqnnvkgfkl nfsgdgssdfsikgdasilkelglsdvnii skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn flagellar hook-65 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejunimninvtqlaq kdvyqskglandggfvnaql CF93-6] ngtadltffs ngkeytvtvd knttyrdladGenBank: kineasggei vakivntgekgtpyrltlts ketgedsais EAQ57731.1fyagkkdsng kyqkdinaek ifddlgwgld vsasidpdkdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn Flagellar hook-66 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni 4031]mninvtqlaq kdvyqsqglandggfvnakl GenBank:ngtadltffs ngkeytvtvd knttyrdlad CDH62398.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyksdseaek ifkslgweld ttssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn Flagellar hook-67 mafgslsslg fgsgvltqdt idklkeaeqk associated protein 2aridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni M1]mninvtqlaq kdvyqskglandsgfvnanl GenBank:tgttdltffs ngkeytvtvd knttyrdlad ADN90737.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyqsdpeaen ifsnlgweld kttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpd stesffsnitkyedinhtge vikqgslnqyldssgtgnkg ldfkpgdfti vfnnqtydls knsdgtnfkltgkteeellq nlanhinskgieglkvkves ydqngvkgfk lnfsgdgssdfsikgnatilqelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar filament cap 68 mafgslsslg fgsgvltqdt idklkeaeqk protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni str.mninvtqlaq kdvyqskglandsgfinanl RM3420]tgttdltffs ngkeytvtvd ksttyrdlad kineasggei GenBank:vakivntgekgtpyrltlts ketgedsais AOW96893.1fyagkkdaqg qyksdseaek ifsnlgweld kttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpd stesffsnitkyedinhtge vikqgslnqyldssgtgnkg lefkpggfti vfnnqtydls knsdgtnfkltgkteeellq nlanhinskgieglkvkves ydqngvkgfk lkfsgdgssdfsikgdanilkelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar filament cap 69 mafgslsslg fgsgvltqdt idklkeaeqk protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni]mninvtqlaq kdvyqsqglandggfvnakl GenBank:ngtadltffs ngkeytvtvd knttyrdlad AON66729.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyksdpeaek ifkslgweld ttssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgikgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament cap 70 mafgslsslg fgsgvltqdt idklkeaeqk protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni]mninvtqlaq kdvyqsqglandsgfinanl GenBank:agttdltffs ngkeytvtvd knttyrdlad AON65179.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyqsdpeaek ifsnlgweld kttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpd stesffsnitkyedinhtge viktgslskylnsnggntng lafkpgdfti vfnnqtydls knsdgtnfkltgkteeellq nlanhinskgieglkvkves ynqnnvtgfr lnfsgdgssdfsikgdgsilkelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar filament cap 71 mafgslsslg fgsgvltqdt idklkeaeqk proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni]mninvtqlaq kdvyqskglandggfvnaql GenBank:ngtadltffs ngkeytvtvd knttyrdlad AOH51565.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdseaen ifsnlgweld ktssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev intgslskylnpngldfkpg dftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhi nskgieglkvkvesynqnnv tgfrinfsgd gssdfsikgn atilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtrydtmanqwlq yesilnklnqqlntvtnmin aannsnn flagellar filament cap 72mafgslsslg fgsgvltqdt idklkeaeqk protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni]mninvtqlaq kdvyqskglandggfvnaql GenBank:ngtadltffs ngkeytvtvd knttyrdlad ALF93210.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng kyqkdtnaek ifddlgwgld asasidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlnfdsskf eqkvkedpdsaesffsnitkyedinhtgei iktgnlskyl nsnggntngl dfkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy dqnnvkgfkl nfsgdgssdfsikgdasilkelglsdvnii skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn Flagellar hook-73 mafgslsslg fgsgvltqdt idklkeaeqk associated protein 2aridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni]mninvtqlaq kdvyqsqglandggfvnaql GenBank: AJP35034.1ngtadltffs ngkeytvtvd knttyrdladkineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyesdseaek ifkslgweld ttssinpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efqpgnftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy dqngvkgfrl nfsgdgssdfsikgdanilkdlglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament cap 74 mafgslsslg fgsgvltqdt idklkeaeqk proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni]mninvtqlaq kdvyqskglandggfvnaql GenBank:ngtadltffs ngkeytvtvd knttyrdlad AOH51565.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdseaen ifsnlgweld ktssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev intgslskylnpngldfkpg dftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhi nskgieglkvkvesynqnnv tgfrinfsgd gssdfsikgn atilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtrydtmanqwlq yesilnklnqqlntvtnmin aannsnn flagellar capping 75 mafgslsslg fgsgvltqdt idklkeaeqkprotein aridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni CG8421]mninvtqlaq kdvyqsqglandsgfinanl GenBank:agttdltffs ngkeytvtvd knttyrdlad AHY39787.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyksdleaek ifkslgweld ttssidpakdkkgygikdas lhiqtagnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl afkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdgsilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar cap protein 76 mafgslsslg fgsgvltqdt idklkeaeqkFliD [Campylobacter aridpytkki eenttkqkdl teiktkllsfqtavssladajejuni 32488] tvfakrkvvg sisdnppasl tvnsgvalqs GenBank:mninvtqlaq kdvyqsqglandggfvnakl AGQ95247.1ngtadltffs ngkeytvtvd knttyrdladkineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdvqg qyksdseaek ifkslgweld ttssidpakd kkgygikdas lhiqtagnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar capping 77 mafgslsslg fgsgvltqdt idklkeaeqk proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter jejunitvfakrkvvg sisdnppasl tvnsgvalqs subsp. jejuni IA3902]mninvtqlaq kdvyqskglandggfvnaql GenBank:ngtadltffs ngkeytvtvd knttyrdlad ADC28162.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng kyqkdinaek ifddlgwgld vsasidpdkdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn flagellar hook-78 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni subsp. jejuni]mninvtqlaq kdvyqskglandsgfvnanl 81116 tgttdltffs ngkeytvtvd knttyrdladGenBank: kineasggei vakivntgekgtpyrltlts ketgedsais ABV52108.1fyagkkdaqg qyqsdpeaen ifsnlgweld kttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpd stesffsnitkyedinhtge vikqgslnqyldssgtgnkg ldfkpgdfti vfnnqtydls knsdgtnfkltgkteeellq nlanhinskgieglkvkves ydqngvkgfk lnfsgdgssdfsikgnatilqelglsdvni tskpiegkgi fsklkatlqe mtgkdgsitk ydesltndikslntskdstqamidtrydtm anqwlqyesi lnklnqqlnt vtnminaann snnflagellar hook- 79 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs jejuni RM1221]mninvtqlaq kdvyqskglandsgfinanl GenBank:tgttdltffs ngkeytvtvd knttyrdlad AAW35835.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdaqg qyksdseaee ifkslgweld tassidpakd kkgygikdps lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl tnglefkpgdftivfnnqty dlsknsdgtn fkltgkteeellqnlanhinskgieglkvk vesynqnnvt gfrinfsgdgssdfsikgna silkelglsdvnitskpieg kgifsklkatlqemtgkdgs itkydesltn dikslntskd stqamidtrydtmanqwlqy esilnklnqqlntvtnmina annsnn flagellar cap protein 80mafgslsslg fgsgvltqdt idklkeaeqk FliD [Campylobacteraridpytkki eenttkqkdl teiktkllsfqtavsslada coli RM5611]tvfakrkvvg sisdnppasl tvnsgvalqs GenBank:mninvtqlaq kdvyqskglandsgfinanl AHK75426.1tgttdltffs ngkeytvtvd knttyrdladkineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdseaen ifsnlgweld ktssidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgnasilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar cap protein 81 mafgslsslg fgsgvltqdt idklkeaeqkFliD [Campylobacter arinpytkki eenttkqkdl teiktkllsfqtavssladacoli RM4661] tvfakrkvvg sisdnppasl tvnsgvalqn GenBank:mninvtqlaq kdvyqskglandsgfvnaql AHK76446.1ngtadltffs ngkeytvtvd knttyrdlad kineasggei vakivntgekgapyrltltsketgedsais fyagkkdssg kytsdsnaet iflmlgweld ttssidpdkdkkgygikdaslhiqtaqnae ftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtn lnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedvngnkvntkvmlsm qdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgevintgslskyl npngldfkqg dftivfnnqt ydlsknsdgt nflthgkteeellqnlanhinskgieglkv kvesynqngv kgfklnfsgdgssdfsikgn asilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtr ydtmanqwlq yesilnklnqqlntvtnmin aannssn Flagellar hook- 82 mafgslsslg fgsgvltqdt idklkeaeqkassociated protein aridpytkki eenttkqkdl teiktkllsfqtavssladaFliD [Campylobacter tvfakrkvvg sisdnppasl tvnsgvalqs coli 15-537360]mninvtqlaq kdvyqskglandggfvnaql GenBank:ngtadltffs ngkeytvtvd knttyrdlad AGZ21001.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdseaen ifsnlgweld ktssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdanilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 83 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter coli] tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglyndggfvnaql Sequence:ngtadltffs ngkeytvtvd knttyrdlad WP_004284951.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng kyqkdtnaek ifddlgwgld vsasidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfnsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnglefkpgdftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhinskgieglkv kvesynqnnv tgfrinfsgdgssdfsikgn asilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtr ydtmanqwlq yesilnklnqqlntvtnmin aannsnn Flagellar hook- 84 mafgslsslg fgsgvltqdt idklkeaeqkassociated protein 2 aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter coli] tvfakrkvvg sisdnppasl tvnsgvalqs GenBank:mninvtqlaq kdvyqskglandggfvnaql AJW57994.1ngtadltffs ngkeytvtvd knttyrdladkineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng kyqkdtnaek ifddlgwgld vsasidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfnsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnglefkpgdftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhinskgieglkv kvesynqnnv tgfrinfsgdgssdfsikgn asilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtrydtmanqwlq yesilnklnqqlntvtnmin aannsnn Flagellar hook- 85 mafgslsslg fgsgvltqdt idklkeaeqkassociated protein 2 aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter coli] tvfakrkvvg sisdnppasl tvnsgvalqs GenBank:mninvtqlaq kdvyqskglandggfvnaql ALV00075.1ngtadltffs ngkeytvtvd knttyrdladkineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng kytsdseaet ifloilgweld kttqtidpakdkkgygikda slhiqtaqnaeftldgikmf rssntvtdlg vgmtltlnkt geinfdvqqdfegvtkamqd lvdayndlvtnlnaatdyns etgtkgtlqg isevnsirss iladlfdsqvvdgttedang nkvntkvmlsmqdfglslnd agtlsfdssk feqkvkedpdstesffsnitkyedinhtge viktgslsky lnsnglefkp gdftivfnnq tydlsknsdgtnfkltgkteeellqnlanh inskgieglk vkvesynqnn vtgfrinfsg dgssdfsikgnasilkelglsdvnitskpi egkgifsklk atlqemtgkdgsitkydesltndikslntskdstqamidtrydtmanqw l qyesilnkln qqlntvtnmi naannsnnFlagellar hook- 86 mafgslsslg fgsgvltqdt idklkeaeqk associated proteinaridpytkki eenttkqkdl teiktkllsfqtavsslada FliD [Campylobactertvfakrkvvg sisdnppasl tvnsgvalqs coli IPSID-1]mninvtqlaq kdvyqskglandsgfinanl GenBank:tgttdltffs ngkeytvtvd knttyrdlad CDL88777.1kineasggei vakivntgekgtpyrltlts ketgedsais fyagkkdsngkytsdseaetifloilgweldttssidpakdkkgygikdas lhiqtaqnae ftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtn lnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangnkvntkvmlsm qdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgevikqgslnqyl dssgtgnkgl dfkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy dqngvkgfklnfsgdgssdf sikgnatilkelglsdvnit skpiegkgifsklkatlqem tgkdgsitky desltndiks lntskdstqamidtrydtma nqwlqyesilnklnqqlntv tnminaanns nn flagellar hook- 87mafgslsslg fgsgvltqdt idklkeaeqk associated protein 2aridpytkki eenttkqkdl teiktkllsfqtavsslada (fliD), putativetvfakrkvvg sisdnppasl tvnsgvalqs [Campylobacter colimninvtqlaq kdvyqskglandggfvnaql RM2228] ngtadltffs ngkeytvtvd knttyrdladGenBank: kineasggei vakivntgekgtpyrltlts ketgedsais EAL57379.1fyagkkdsng kyqkdtnaek ifddlgwgld vsasidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfnsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnglefkpgdftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhinskgieglkv kvesynqnnv tgfrinfsgdgssdfsikgn asilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtrydtmanqwlq yesilnklnqqlntvtnmin aannsnn flagellar filament 88mafgslsslg fgsgvltqdt idklkeaeqk capping protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter coli]tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglandggfvnaql Sequence:ngtadltffs ngkeytvtvd knttyrdlad WP_002842748.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng kyqkdtnaek ifddlgwgld vsasidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnglefkpgdftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhinskgieglkv kvesynqnnv tgfrinfsgdgssdfsikgn asilkelglsdvnisskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtrydtmanqwlq yesilnklnqqlntvtnmin aannsnn flagellar filament 89mafgslsslg fgsgvltqdt idklkeaeqk capping protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter coli]tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglandggfvnaql Sequence:ngtadltffs ngkeytvtvd knttyrdlad WP_002833936.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdseaen ifsnlgweld ktssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfnsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnglefkpgdftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhinskgieglkv kvesynqnnv tgfrinfsgdgssdfsikgn asilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtrydtmanqwlq yesilnklnqqlntvtnmin aannsnn flagellar hook- 90 mafgslsslg fgsgvltqdt idklkeaeqkassociated protein 2 aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter coli] tvfakrkvvg sisdnppasl tvnsgvalqs JV20mninvtqlaq kdvyqskglyndggfvnaql GenBank:ngtadltffs ngkeytvtvd knttyrdlad EFM36457.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng kyqkdtnaek ifddlgwgld vsasidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfnsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnglefkpgdftivfnnqt ydlsknsdgt nfkltgkteeellqnlanhinskgieglkv kvesynqnnv tgfrinfsgdgssdfsikgn asilkelglsdvnitskpie gkgifsklkatlqemtgkdg sitkydeslt ndikslntsk dstqamidtrydtmanqwlq yesilnklnqqlntvtnmin aannsnn flagellar filament 91mafgslsslg fgsgvltqdt idklkeaeqk capping protein FliDaridpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter coli]tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglandggfvnaql Sequence:ngtadltffs ngkeytvtvd knttyrdlad WP_002832776.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdsgaen ifsnlgweld ktssidpakdkkgygikdas lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpds tesffsnitkyedinhtgev iktgnlskylnsnggntngl efkpgdftiv fnnqtydlsk nsdgtnfkltgkteeellqnlanhinskgieglkvkvesy nqnnvtgfrl nfsgdgssdf sikgdanilkelglsdvnit skpiegkgifsklkatlqem tgkdgsitky desltndiks lntskdstqamidtrydtma nqwlqyesilnklnqqlntv tnminaanns nn flagellar filament 92mafgslsslg fgsgvltqdt idklkeaeqk capping protein FliDarinpytkki eenttkqkdl teiktkllsfqtavsslada [Campylobacter coli]tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglandsgfinanl Sequence:tgttdltffs ngkeytvtvd ksttyrdlad kineasggei WP_002825071.1vakivntgekgtpyrltlts ketgedsais fyagkkdssg kytsdsnaet ifloilgweldttssidpdkdkkgygikdas lhiqtaqnae ftldgikmfr ssntvtdlgv gmtltlnktgeinfdvqqdfegvtkamqdlvdayndlvtn lnaatdynse tgtkgtlqgi sevnsirssiladlfdsqvvdgttedvngn kvntkvmlsm qdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev intgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgnasilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 93 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter coli] tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglandsgfinanl Sequence:tgttdltffs ngkeytvtvd knttyrdlad WP_002804771.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdseaen ifsnlgweld ktssidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgnasilkelglsdvnit skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 94 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter coli] tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglandsgfvnanl Sequence:tgttdltffs ngkeytvtvd knttyrdlad WP_002793506.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdsng qyqsdseaen ifsnlgweld ktssidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdlvdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqnlanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdasilkelglsdvnis skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nnflagellar filament 95 mafgslsslg fgsgvltqdt idklkeaeqkcapping protein FliD aridpytkki eenttkqkdl teiktkllsfqtavsslada[Campylobacter coli] tvfakrkvvg sisdnppasl tvnsgvalqs NCBI Referencemninvtqlaq kdvyqskglandsgfvsanl Sequence:tgttdltffs ngkeytvtvd knttyrdlad WP_002791831.1kineasggei vakivntgekgtpyrltlts ketgedsaisfyagkkdssg kytsdsnaet ifloilgweld ktssidpakdkkgygikdts lhiqtaqnaeftldgikmfr ssntvtdlgv gmtltlnktg einfdvqqdfegvtkamqdl vdayndlvtnlnaatdynse tgtkgtlqgi sevnsirssi ladlfdsqvvdgttedangn kvntkvmlsmqdfglslnda gtlsfdsskf eqkvkedpdstesffsnitkyedinhtgev iktgslskyl nsnggntngl efkpgdftiv fnnqtydlsknsdgtnfkltgkteeellqn lanhinskgi eglkvkvesy nqnnvtgfrl nfsgdgssdfsikgdasilkelglsdvnis skpiegkgif sklkatlqem tgkdgsitky desltndikslntskdstqamidtrydtma nqwlqyesil nklnqqlntv tnminaanns nn

Example 3 Campylobacter-Specific Antibodies in rSIgA2 Bind to FliD

In the SIgA2 format, CAA1 and CCG4 maintained the original FliD bindingactivity, displaying similar EC50 for the flagellar protein (FIG. 7A).The EC50 of CAA1 SIgA2 was 0.02372, and the EC50 of CCG4 SIgA2 was0.02954. Epitope binning showed that the binding of one antibody to theantigen did not prevent the binding of the other mAb (FIG. 7B), thusindicating the recognition of two different epitopes. Western blotanalysis in denaturing reducing conditions indicated that the two mAbsbind structurally different antigenic determinants, with CAA1 targetinga linear epitope and CCG4 a conformational one (FIG. 7C). Theaccessibility of the antibodies to these epitopes in the flagella of C.jejuni and C. coli historical isolates was confirmed by flow cytometryand by confocal imaging (FIGS. 7D, 7E).

Example 4 rSIgA Inhibits Motility of Campylobacter In Vitro

A motility assay is shown schematically in Figure. 2. Results areprovided in FIG. 3. Binding of CAA1 (rSIgA2) to the Campylobacterflagella reduced bacteria motility, a condition required to maximizeinfection and invasiveness. The control rSIgA2 HGN194, directed againstHIV-1 gp120, was unable to curb bacteria diffusion in the motilityassay, indicating that that the inhibition of bacterial motility was notresulting from “innate” cross-reactivity of the highly glycosylatedstructure of SIgA.

Example 5 Mouse Model of Campylobacter Infection

Genetically manipulated animals characterized by an exacerbatedinflammatory responses to bacteria, such as SIGIRR or IL10⁻/⁻ mice, havebeen proposed as models to study Campylobacter pathogenesis (Heimesaatet al., Front. Cell. Infect. Microbiol., 4:77 (2014); Stahl et al., PLoSPathog., 10:e1004264 (2014)). However, these mutations dramaticallyalter the murine immune system to an extent that even the presence ofcommensal microbes can potentially result in spontaneous enterocolitis(Mansfield et al., Infect. Immun., 75:1099-1115 (2007)).

Moreover, the murine intestine has been shown to be highly resistant toCampylobacter due to colonization resistance and a certain level oftolerance, which limits inflammation (Bereswill et al., PLoS One,6:e20953 (2011); Chang and Miller, Infect. Immun., 74:5261-5271 (2006);Masanta et al., Clin. Dev. Immunol., 2013:526860 (2013)). To overcomethe potential effects of the resident microbiota, pre-treatment via oralgavage with vancomycin, for which Campylobacter species are inherentlyresistant (Taylor and Courvalin, Antimicrob. Agents Chemother.,32:1107-1112 (1988)), was adopted. Although the pretreatment allowsrobust bacterial colonization in the caecum, it does not appear toenhance the pathology in adult wild type mice, as minimal signs ofinflammation were observed (Stahl et al., PLoS Pathog., 10:e1004264(2014)).

Higher susceptibility to C. jejuni infection of infant wild type mice incomparison to adult animals has been previously reported and linked tosignificant differences in the microbiota composition (Haag et al., Eur.J. Microbiol. Immunol. (Bp), 2:2-11 (2012)). To set up a model thatcould recapitulate the disease in newborn and infants underimmunocompetent conditions, the sensitivity to C. jejuni 81-176infection of was evaluated in pups (12 day-old), just-weaned (21day-old) and adult (56 day-old) C57BL/6 mice. Animals were pre-treatedwith vancomycin via oral gavage to deplete the murine microbiota beforebeing infected with C. jejuni 81-176 at 10⁹ CFU/mouse. Campylobacterisolation from stools at 6 days post-infection revealed almost2-log-higher shedding from 21-day-old animals than from 12 and56-day-old mice (FIG. 8A). In-line with these results, just-weaned micedisplayed significantly higher intestinal inflammation as measured bythe levels of lipocalin-2 in the stools and histological score values inthe caecum in comparison to pups and adult mice (FIGS. 8B-8C).

Since the antibiotic pre-treatment is expected to provide comparableecological niches for infection in the different animals, other factorscould account for the different sensitivity to C. jejuni infectiondisplayed by the three groups of mice. Analysis of murine IgA in thestools of 12, 21 and 56-day-old mice revealed different concentrationsamong the groups (FIG. 8D). Notably, just-weaned mice presentednegligible levels of IgA in the stools in comparison to both pups andadult mice. Without wishing to be bound by theory, this observation maybe consistent with the transition between the exogenous supply ofmaternal antibodies provided with milk (12 days-old mice) and thebeginning of the endogenous production that is established in adultanimals (56 days-old mice). Therefore, a factor in the susceptibility ofjust-weaned mice to C. jejuni infection may be a lower concentration ofsecretory IgA due to the relative immaturity of intestinal immune systemand the depletion of maternal antibodies in these animals. Based onthese data, just-weaned mice were selected as an immune competent mousemodel to study the prophylactic activity of FliD-reactive mAbs.

Off-target binding by CAA1 and CCG4 to the murine microbiota couldresult in reduced mAb availability and thus, reduced activity againstpathogens in a prophylactic setting. To investigate this, potentialcross-reactivity of the rSIgAs with the microbiota of just-weaned micewas evaluated. Stools from animals orally infected with C. jejuni, C.coli or PBS (mock infected) were collected 24 hours post-infection andincubated with the two FliD-reactive mAbs and the control rSIgA HGN194.Analysis of human-IgA coated bacteria from stools of mock and infectedanimals revealed that both Campylobacter-reactive rSIgA were able torecognize and bind the most common species associated with severeinfections, displaying limited cross-reactivity with the murinemicrobiota (FIG. 14A).

To set-up the conditions for testing the prophylactic efficacy of theantibodies, the pharmacokinetics of orally administered SIgA indifferent gastrointestinal tracts of just weaned mice were evaluated.The Campylobacter-irrelevant HGN194 rSIgA2, which displayed nocross-reactivity with the murine microbiota (FIG. 14A), was administeredas a single oral gavage of 150 ug in PBS (≈15 mg/Kg) and itsconcentration in the different intestinal sub-compartments was measuredat 2, 4 and 8 h post-administration (FIG. 14B). HGN194 rSIgAconcentration in the caecum was maintained almost constant within thefirst 4 hours post-administration and then tended to dramaticallydecrease by 8 hours (FIG. 14B). The human antibody was not detectable at12- or 24-hours post-administration (data not shown).

Example 6 Prophylactic Effect of Orally Administered rSIgA

Just-weaned animals (21d) were treated with vanocmycin and thenadministered a single oral gavage of 200 μg/mouse of rSIgA2 CAA1, CCG4,HGN194 or PBS two hours before oral infection with 10⁸ CFU/mouse of C.jejuni 81-176. Treated animals and the corresponding control groups weremonitored for 72 hours, during which the severity of infection anddegree of inflammation were recorded. Analysis of the stools fromtreated mice revealed a trend characterized by higher Campylobactershedding at 24 hours post-infection followed by a significant decreaseover time. Conversely, untreated and HGN194-treated groups presentedlower shedding at early time points followed by a consistent CFUincrease at 48 hours post-infection (FIG. 9A). FIGS. 5A and 5B show datafrom a separate experiment in which mice were administered 200 μg mAbonce before infection, and twice after infection, with 10⁹ CFU/mouse ofC. jejuni 81-176.

These results suggest that CAA1 and CCG4 may prevent or reduce theability of the pathogen to adhere to the surface of the mucosalepithelium, thus facilitating the clearance of bacteria via peristalsisor mucocilliary activity at early stages post-infection. This hypothesiswas further supported by significantly lower levels of lipocalin-2, amarker of intestinal inflammation linked to epithelial damage andneutrophil infiltration, recorded at 72 hours post-infection in thestools of CAA1 and CCG4 treated animals in comparison to the controlgroups (infected/non-treated and infected/HGN194 treated groups) (FIG.9B). FIG. 6 shows data from a separate experiment in which mice wereadministered 200 μg mAb once before infection, and twice afterinfection, with 10⁹ CFU/mouse of C. jejuni 81-176.

Similar findings were observed in animals administered with higher orlower Campylobacter inoculum (10⁷ or 10⁹ CFU/mouse; FIGS. 15A, 15B). Inaddition, animals treated with a single administration of FliD-reactivemAbs presented levels of PMN cells infiltration and histological scorevalues in the caecum that were comparable with non-infected mice andsignificantly lower than the HGN194-treated group, hence supporting invivo efficacy that is not driven by the “innate activity” (Kaetzel,Immunol. Rev., 206:83-99 (2005); Phalipon et al., Immunity, 17:107-115(2002)), associated with the highly glycosylated SIgA (FIGS. 9C, 9D).

These results indicate that FliD-specific antibodies of the presentdisclosure in rSIgA2 format protect against Campylobacter infection andinflammation following oral delivery by accelerating bacterial clearanceat early stages after infection.

Example 7 IgA Isotype Switch does not Affect CAA1 Prophylactic Activity

Since IgA1 and IgA2 can have differences in Fab reach, flexibility, andglycosylation that might affect the cross-linking ability and/orpersistence of the polymeric Ig in the intestine, the followingexperiments were performed to determine whether the two IgA isotypes mayexert different prophylactic activities in the herein-describedimmunocompetent mouse model of Campylobacter infection.

FliD-reactive CAA1 was recombinantly produced as SIgA1 and SIgA2. Properassembly of the two subclasses was confirmed by analytical methods andby digestion with IgA1 proteases from Neisseria gonhorrehoeae (FIG.16A). CAA1 SIgA1 and SIgA2 displayed comparable binding to FliD (FIG.16B) and no significant differences in reactivity to Campylobacterspecies in vitro or with murine microbiota ex vivo were observed betweenthe two formats (FIGS. 16C, 16D).

The prophylactic activity of the two subclasses was then tested in themurine model of Campylobacter infection. In line with previous findings,animals administered the FliD-reactive mAbs displayed higherCampylobacter shedding at early timepoints post-infection followed by adecrease over time, while infected non-treated animals produced anopposite trend (FIG. 10A). Although CAA1 rSIgA2 accelerated sheddingfaster than rSIgA1, both subclasses were equally capable of limitinginflammation in infected animals, as shown by the levels of lipocalin-2in the stools, the PMN infiltration and the corresponding histologicalscore in the caecum at 72 hours post-infection (FIGS. 10B-10D).

These results indicate that structural differences between IgA1 and IgA2do not result in differences in prophylactic activity exerted by thesetwo CAA1 formats in the in-vivo model.

Example 8 mAbs CAA1 and CCG4 have Reduced Oral Prophylactic Activitywhen Expressed as IgG

Although SIgAs are thought to be the most abundant antibodies expressedin association with the intestinal mucosa and may be the first line ofdefense against enteric pathogens, they are characterized by a complexprotein structure and their development as drugs may present challengesin comparison to IgG-based monoclonal antibodies. Since the activity ofthe Campylobacter-reactive mAbs was shown to be dependent on specificityfor FliD, CAA1, CCG4 and the Campylobacter-irrelevant antibody HGN194were generated as rIgG1 and evaluated for prophylactic activity incomparison to their corresponding SIgA2 counterparts.

Since glycosylation might affect the ability of mAbs to interact withmucin on the mucosal surface, the localization and persistence ofcontrol mAb HGN194 as rIgG1 in the murine intestinal tract was appraisedby administering the antibody be a single oral gavage to 21-day old miceand then by measuring its concentration in the different intestinalsub-compartments after 2, 4 and 8 h (FIG. 14C). As with HGN194 rSIgA2,at every time-point, the highest concentration of the rIgG1 was detectedin the caecum; however, in this intestinal sub-compartment the rIgG1concentration tended to decrease faster than rSIgA2, with a significantreduction observed by 4 hours post-administration (FIGS. 14B, 14C).

The prophylactic activity of the FliD-reactive mAbs CAA1 and CCG4 asrIgG1 or SIgA2 was also evaluated. MAbs were administered orally tojust-weaned mice 2 hours before infection with C. jejuni 81-176.Interestingly, while animals treated with SIgA2 antibodies displayed thepreviously observed pattern characterized by higher shedding at 24 hourspost-infection followed by a significant decrease at 48 and 72 h, thegroups treated with the IgG version of the same antibodies revealedtrends similar to the non-treated groups (FIGS. 11A and 17A). Theimportance of the SIgA format for in vivo efficacy was further confirmedby the lower ability of CAA1 and CCG4 IgG to limit inflammation incomparison to their polymeric counterparts, as shown by PMN cellsinfiltration in the caecum and lipocalin-2 levels in the stools of theinfected animals at 72 post-infection (FIGS. 11B, C and 17B, 17C).Overall, no significant differences in the histological scores wereobserved between the mice treated with the FliD-reactive IgG antibodiesand the non-treated animals. Conversely, the SIgA versions of theantibodies were able to replicate the beneficial effect previouslyobserved, maintaining the histological score in the caecum to valuessignificantly lower than both non-treated and IgG treated mice (FIGS.11D and 17D).

These data indicate that CAA1 and CCG4 IgGs have limited prophylacticactivity when orally administered prior to Campylobacter infection, ascompared to the same antibodies expressed as SIgA. Without wishing to bebound by theory, the lack of activity of orally administered CAA1 andCCG4 IgGs might rely both on a lower persistence in the gastrointestinaltract and on different cross-linking properties associated with the SIgAformat.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, includingU.S. Provisional Patent Application No. 62/699,573, filed Jul. 17, 2018,are incorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. An isolated antibody, or an antigen-bindingfragment thereof, that is specific for a Campylobacter flagellum cappingprotein (FliD) epitope.
 2. The antibody, or an antigen-binding fragmentof claim 1, wherein the epitope is a conformational epitope.
 3. Theantibody, or an antigen-binding fragment of claim 1, wherein the epitopeis a linear epitope.
 4. The antibody or antigen-binding fragment of anyone of claims 1-3, wherein the antibody or antigen-binding fragment iscapable of binding to the FliD epitope with an EC50 of less than about0.1 μg/mL, or less than about 0.05 μg/mL, or less than about 0.03 μg/mL,as measured by ELISA.
 5. The antibody or antigen-binding fragment of anyone of claims 1-4, wherein the antibody or antigen-binding fragment iscapable of reducing motility of the Campylobacter in an in vitro cellmotility assay.
 6. The antibody or antigen-binding fragment of any oneof claims 1-5, wherein the antibody or antigen-binding fragment iscapable of neutralizing a Campylobacter infection in a subject.
 7. Theantibody or antigen-binding fragment of any one of claims 1-6, whereinthe Campylobacter comprises Campylobacter jejuni, Campylobacter coli, orboth.
 8. The antibody or antigen-binding fragment of any one of claims1-7, wherein the Campylobacter comprises C. jejuni 81-176, C. coli10092/ATB, or both.
 9. The antibody or antigen-binding fragment of anyone of claims 1-8, wherein the antibody or antigen-binding fragmentcomprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acidsequences according to: (i) SEQ ID NOs:9-14, respectively; or (ii) SEQID NOs:25-30, respectively.
 10. The antibody or antigen-binding fragmentof any one of claims 1-9, wherein the antibody or antigen-bindingfragment comprises at least one more germline-encoded amino acid in avariable region as compared to a parent antibody or antigen bindingfragment, provided that the parent antibody or antigen binding fragmentcomprises one or more somatic mutations.
 11. The antibody orantigen-binding fragment of any one of claims 1-10, wherein the antibodyor antigen-binding fragment comprises: (i) VH having at least 85% aminoacid identity to SEQ ID NO:2, and a VL having at least 85% amino acididentity to SEQ ID NO:4; or (ii) VH having at least 85% amino acididentity to SEQ ID NO:22, and a VL having at least 85% amino acididentity to SEQ ID NO:24.
 12. The antibody or antigen-binding fragmentof claim 11, wherein the antibody or antigen-binding fragment comprises:(i) a VH according to SEQ ID NO:2, and a VL according to SEQ ID NO:4; or(ii) a VH according to SEQ ID NO:22, and a VL according to SEQ ID NO:24.13. The antibody or antigen-binding fragment of any one of claims 1-12,wherein the antibody or antigen-binding fragment is an IgA, IgG, IgD,IgE, or IgM isotype.
 14. The antibody or antigen-binding fragment ofclaim 13, wherein the antibody or antigen-binding fragment is an IgAisotype.
 15. The antibody or antigen-binding fragment of claim 14,wherein the antibody or antigen-binding fragment is an IgA1 isotype. 16.The antibody or antigen-binding fragment of claim 14, wherein theantibody or antigen-binding fragment is an IgA2 isotype.
 17. Theantibody or antigen-binding fragment of any one of claims 1-16, whereinthe antibody or antigen-binding fragment comprises a Fc polypeptide or afragment thereof.
 18. The antibody or antigen-binding fragment of claim17, comprising a heavy chain constant region having at least 90%identity to any one of SEQ ID NOs:40-42.
 19. The antibody orantigen-binding fragment of any one of claims 14-18, wherein theantibody or antigen-binding fragment comprises an IgA dimer molecule.20. The antibody or antigen-binding fragment of any one of claims 14-19,wherein the antibody or antigen binding fragment comprises a secretoryIgA molecule.
 21. The antibody or antigen-binding fragment of any one ofclaims 1-20, wherein the antibody or antigen binding fragment ismonoclonal.
 22. The antibody or antigen-binding fragment of any one ofclaims 1-21, wherein the antibody or antigen binding fragment ischimeric, humanized, or human.
 23. A composition, comprising theantibody or antigen-binding fragment of any one of claims 1-22, and apharmaceutically acceptable carrier, excipient, or diluent.
 24. A kit,comprising: a first antibody or an antigen-binding fragment thereof,which is specific for a Campylobacter flagellum capping protein (FliD)linear epitope; and (ii) a second antibody or an antigen-bindingfragment thereof, which is specific for a Campylobacter flagellumcapping protein (FliD) conformational epitope.
 25. The kit of claim 24,wherein: (i) the first antibody or antigen-binding fragment comprisesHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences according to SEQID NOs:9-14, respectively; and (ii) the second antibody orantigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3 sequences according to SEQ ID NOs:25-30, respectively.
 26. Thekit of claim 24 or 25, wherein: (i) the first antibody orantigen-binding fragment comprises a VH having at least 85% amino acididentity to SEQ ID NO:2, and a VL having at least 85% amino acididentity to SEQ ID NO:4; and (ii) the second antibody or antigen-bindingfragment comprises a VH having at least 85% amino acid identity to SEQID NO:22, and a VL having at least 85% amino acid identity to SEQ IDNO:24.
 27. The kit of claim 26, wherein: (i) the first antibody orantigen-binding fragment comprises a VH according to SEQ ID NO:2, and aVK according to SEQ ID NO:4; and (ii) the second antibody orantigen-binding fragment comprises a VH according to SEQ ID NO:22, and aVL according to SEQ ID NO:24.
 28. The kit of any one of claims 24-27,wherein the first antibody or antigen-binding fragment and the secondantibody or antigen-binding fragment are each a same isotype.
 29. Thekit of claim 28, wherein the first antibody or antigen-binding fragmentand the second antibody or antigen-binding fragment are each a secretedIgA.
 30. An isolated polynucleotide encoding the antibody orantigen-binding fragment of any one of claims 1-22.
 31. The isolatedpolynucleotide of claim 30, wherein the polynucleotide iscodon-optimized for expression in a host cell.
 32. The isolatedpolynucleotide of claim 30 or 31, comprising: (i) a VH-encodingpolynucleotide having at least 75% identity to the nucleotide sequenceset forth in any one of SEQ ID NOs:1, 5, 7, 8, 21, 37, or 38; (ii) aVL-encoding polynucleotide having at least 75% identity to thenucleotide sequence set forth in SEQ ID NO:3, 6, 23, or 39; and/or (iii)HCDR1-, HCDR2-, HCDR3-, LCDR1-, LCDR2-, and LCDR3-encoding sequenceshaving at least 90% identity to the nucleotide sequences set forth inSEQ ID NOs:15-20, respectively, or in SEQ ID NOs:31-36, respectively.33. A vector, comprising the polynucleotide of any one of claims 30-32.34. A recombinant host cell, comprising the isolated polynucleotide ofany one of claims 30-32, or the vector of claim
 33. 35. The recombinanthost cell of claim 34, wherein the host cell comprises a mammalian cell.36. The recombinant host cell of claim 35, wherein the host cell is aCHO cell, a HEK293 cell, a PER.C6 cell, a Y0 cell, a Sp2/0 cell, a NS0cell, a human liver cell, a myeloma cell, or a hybridoma cell.
 37. Amethod of making the antibody or antigen-binding fragment of any one ofclaims 1-22, the method comprising culturing the recombinant host cellof any one of claims 34-36 under conditions and for a time suitable toexpress the antibody or antigen-binding fragment.
 38. A method fortreating a Campylobacter infection in a subject, the method comprisingadministering to the subject an effective amount of the antibody orantigen-binding fragment of any one of claims 1-22, or of thecomposition of claim
 23. 39. A method for reducing intestinalinflammation in a subject having a Campylobacter infection, the methodcomprising administering to the subject an effective amount of theantibody or antigen-binding fragment of any one of claims 1-22, or ofthe composition of claim
 23. 40. A method for increasing intestinalshedding of a Campylobacter by a subject having a Campylobacterinfection, the method comprising administering to the subject aneffective amount of the antibody or antigen-binding fragment of any oneof claims 1-22, or of the composition of claim
 23. 41. The method of anyone of claims 38-40, wherein the antibody or antigen-binding fragmentcomprises a secretory IgA molecule.
 42. The method of any one of claims38-41, wherein the administering comprises oral administration of theantibody, antigen-binding fragment, or composition.
 43. The method ofclaim any one of claims 38-42, wherein the administering comprisesadministering the antibody, antigen-binding fragment, or composition tothe subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.
 44. The methodof any one of claims 38-43, wherein the method comprises administeringthe antibody, antigen-binding fragment, or composition to the subject aplurality of times, wherein a second or successive administration isperformed at about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 24, about 48, about 74, about 96 hours, or more,following a first or prior administration, respectively.
 45. The methodof any one of claims 38-44, wherein the antibody, antigen-bindingfragment, or composition is administered to the subject at least onetime prior to the subject being infected by the Campylobacter.
 46. Themethod of any one of claims 38-45, wherein following the administering,a stool sample from the subject comprises an increased number ofCampylobacter colony-forming units (CFUs) as compared to a stool samplefrom the subject prior to being administered an effective amount of theantibody, antigen-binding fragment, or composition.
 47. The method ofany one of claims 38-46, wherein following the administering, a stoolsample from the subject comprises a reduced amount of lipocalin-2 (LCN2)as compared to a stool sample from the subject prior to beingadministered an effective amount of the antibody, antigen-bindingfragment, or composition.
 48. The method of any one of claims 38-47,wherein following the administering, the subject comprises a reducedamount of polymorphonucleated (PMN) cell infiltrate in a caecum ascompared to the subject prior to being administered an effective amountof the antibody, antigen-binding fragment, or composition, wherein thePMN cells are Gr1⁺CD11b⁺.
 49. The method of any one of claims 38-48,wherein following the administering, the subject has an improved caecumhistology as compared to the subject prior to being administered aneffective amount of the antibody, antigen-binding fragment, orcomposition.
 50. The method of any one of claims 38-49, whereinfollowing the administering, the antibody or antigen-binding fragment ispresent in the caecum and/or in feces of the subject for at least 4hours or for at least 8 hours following the administration.
 51. Theantibody or antigen-binding fragment of any one of claims 1-22, or thecomposition of claim 22, for use in a method of: (a) treating aCampylobacter infection in a subject; (b) reducing intestinalinflammation in a subject having a Campylobacter infection; and/or (c)increasing intestinal shedding of a Campylobacter by a subject having aCampylobacter infection.
 52. The antibody or antigen-binding fragment ofany one of claims 1-22, or the composition of claim 23, for use in amethod of manufacturing or preparing a medicament for: (a) treating aCampylobacter infection in a subject; (b) reducing intestinalinflammation in a subject having a Campylobacter infection; and/or (c)increasing intestinal shedding of a Campylobacter by a subject having aCampylobacter infection.
 53. The antibody or antigen-binding fragment,or composition for use according to claim 52, wherein the medicament isformulated for oral administration.
 54. A weaned non-human mammal that:does not have a mature gastrointestinal immune system; and (iii) has adepleted intestinal flora, wherein the depletion is caused by anantibiotic agent.
 55. The weaned non-human mammal of claim 54, whereinthe weaned non-human mammal further comprises a Camplyobacter infection.56. The weaned non-human mammal of claim 54 or 55, wherein the non-humanmammal is a mouse or a rat.
 57. The weaned non-human mammal of claim 56,wherein the non-human mammal is a mouse of about 18 to about 24 daysold.
 58. The weaned non-human mammal of any one of claims 54-57, whereinthe mammal is recently weaned.
 59. The weaned non-human mammal of anyone of claims 54-58, wherein the antibiotic agent comprises vancomycinor an analog thereof.